BCMA chimeric antigen receptors

ABSTRACT

The invention provides improved compositions for adoptive T cell therapies for B cell related conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application of InternationalPatent Application No. PCT/US/2015/064269, filed Dec. 7, 2015, whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 62/200,505, filed Aug. 3, 2015, and U.S. ProvisionalApplication No. 62/091,419, filed Dec. 12, 2014, where theseapplications are herein incorporated by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BLBD_043_02WO_ST25.txt. The text file is 27 KB,was created on Dec. 4, 2015, and is being submitted electronically viaEFS-Web, concurrent with the filing of the specification.

BACKGROUND

Technical Field

The present invention relates to improved compositions and methods fortreating B cell related conditions. More particularly, the inventionrelates to improved chimeric antigen receptors (CARs) comprising murineanti-BCMA antibodies or antigen binding fragments thereof, immuneeffector cells genetically modified to express these CARs, and use ofthese compositions to effectively treat B cell related conditions.

Description of the Related Art

Several significant diseases involve B lymphocytes, i.e., B cells.Abnormal B cell physiology can also lead to development of autoimmunediseases including, but not limited to systemic lupus erythematosus(SLE). Malignant transformation of B cells leads to cancers including,but not limited to lymphomas, e.g., multiple myeloma and non-Hodgkins'lymphoma.

The large majority of patients having B cell malignancies, includingnon-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), are significantcontributors to cancer mortality. The response of B cell malignancies tovarious forms of treatment is mixed. Traditional methods of treating Bcell malignancies, including chemotherapy and radiotherapy, have limitedutility due to toxic side effects. Immunotherapy with anti-CD19,anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80, and anti-HLA-DRtherapeutic antibodies have provided limited success, due in part topoor pharmacokinetic profiles, rapid elimination of antibodies by serumproteases and filtration at the glomerulus, and limited penetration intothe tumor site and expression levels of the target antigen on cancercells. Attempts to use genetically modified cells expressing chimericantigen receptors (CARs) have also met with limited success. Inaddition, the therapeutic efficacy of a given antigen binding domainused in a CAR is unpredictable: if the antigen binding domain binds toostrongly, the CAR T cells induce massive cytokine release resulting in apotentially fatal immune reaction deemed a “cytokine storm,” and if theantigen binding domain binds too weakly, the CAR T cells do not displaysufficient therapeutic efficacy in clearing cancer cells.

BRIEF SUMMARY

The invention generally provides improved vectors for generating T celltherapies and methods of using the same.

In various embodiments, a chimeric antigen receptor (CAR) is providedcomprising: an extracellular domain that comprises a murine anti-BCMA (Bcell maturation antigen) antibody or antigen binding fragment thereofthat binds one or more epitopes of a human BCMA polypeptide; atransmembrane domain, one or more intracellular co-stimulatory signalingdomains, and a primary signaling domain.

In particular embodiments, the murine anti-BCMA antibody or antigenbinding fragment that binds the human BCMA polypeptide is selected fromthe group consisting of: a Camel Ig, Ig NAR, Fab fragments, Fab′fragments, F(ab)′2 fragments, F(ab)′3 fragments, Fv, single chain Fvantibody (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody,tetrabody, disulfide stabilized Fv protein (“dsFv”), and single-domainantibody (sdAb, Nanobody).

In additional embodiments, the murine anti-BCMA antibody or antigenbinding fragment that binds the human BCMA polypeptide is an scFv.

In some embodiments, the murine anti-BCMA antibody or antigen bindingfragment thereof comprises one or more CDRs as set forth in any one ofSEQ ID NOs: 1-3.

In particular embodiments, the murine anti-BCMA antibody or antigenbinding fragment thereof comprises one or more CDRs as set forth in anyone of SEQ ID NOs: 4-6.

In certain embodiments, the murine anti-BCMA antibody or antigen bindingfragment thereof comprises a variable light chain sequence as set forthin SEQ ID NO: 7.

In particular embodiments, the variable light chain sequence comprisesthe CDR sequences set forth in SEQ ID NOs: 1-3.

In other embodiments, the murine anti-BCMA antibody or antigen bindingfragment thereof comprises a variable heavy chain sequence as set forthin SEQ ID NO: 8.

In additional embodiments, the variable heavy chain sequence comprisesthe CDR sequences set forth in SEQ ID NOs: 4-6.

In further embodiments, the transmembrane domain is from a polypeptideselected from the group consisting of: alpha, beta or zeta chain of theT-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154,and PD1.

In some embodiments, the transmembrane domain is from a polypeptideselected from the group consisting of: CD8α; CD4, CD45, PD1, and CD154.

In certain embodiments, the transmembrane domain is from CD8α.

In particular embodiments, the one or more co-stimulatory signalingdomains are from a co-stimulatory molecule selected from the groupconsisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM),CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223(LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10,LAT, NKD2C SLP76, TRIM, and ZAP70.

In particular embodiments, the one or more co-stimulatory signalingdomains are from a co-stimulatory molecule selected from the groupconsisting of: CD28, CD134, and CD137.

In additional embodiments, the one or more co-stimulatory signalingdomains are from a co-stimulatory molecule selected from the groupconsisting of: CD28, CD134, and CD137.

In additional embodiments, the one or more co-stimulatory signalingdomains is from CD28.

In particular embodiments, the one or more co-stimulatory signalingdomains is from CD134.

In other embodiments, the one or more co-stimulatory signaling domainsis from CD137.

In certain embodiments, a CAR comprises a hinge region polypeptide.

In further embodiments, the hinge region polypeptide comprises a hingeregion of CD8α.

In some embodiments, a CAR comprises a spacer region.

In additional embodiments, the spacer region polypeptide comprises CH2and CH3 regions of IgG1 or IgG4.

In particular embodiments, a CAR comprises a signal peptide.

In further embodiments, the signal peptide comprises an IgG1 heavy chainsignal polypeptide, granulocyte-macrophage colony stimulating factorreceptor 2 (GM-CSFR2) signal peptide, or a CD8α signal polypeptide.

In one embodiment, a CAR comprises an amino acid sequence as set forthin SEQ ID NO: 9.

In various embodiments, a polynucleotide encoding a CAR contemplatedherein, is provided.

In various particular embodiments, a polynucleotide encoding a CAR isprovided, wherein the polynucleotide sequence is set forth in SEQ ID NO:10.

In various certain embodiments, a vector comprising a polynucleotideencoding a CAR contemplated herein or as set forth in SEQ ID NO: 10 isprovided.

In certain embodiments, the vector is an expression vector.

In additional embodiments, the vector is an episomal vector.

In particular embodiments, the vector is a viral vector.

In further embodiments, the vector is a retroviral vector.

In other embodiments, the vector is a lentiviral vector.

In one embodiment, a vector encoding a BCMA CAR comprises thepolynucleotide sequence set forth in SEQ ID NO: 36.

In additional embodiments, the lentiviral vector is selected from thegroup consisting essentially of: human immunodeficiency virus 1 (HIV-1);human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus;caprine arthritis-encephalitis virus (CAEV); equine infectious anemiavirus (EIAV); feline immunodeficiency virus (FIV); bovine immunedeficiency virus (BIV); and simian immunodeficiency virus (SIV).

In particular embodiments, a vector comprises a left (5′) retroviralLTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap(cPPT/FLAP), a retroviral export element; a promoter operably linked tothe polynucleotide encoding a CAR contemplated herein; and a right (3′)retroviral LTR.

In other embodiments, a CAR comprises a heterologous polyadenylationsequence.

In some embodiments, a CAR comprises a hepatitis B virusposttranscriptional regulatory element (HPRE) or woodchuckpost-transcriptional regulatory element (WPRE).

In certain embodiments, the promoter of the 5′ LTR is replaced with aheterologous promoter.

In further embodiments, the heterologous promoter is a cytomegalovirus(CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or an Simian Virus40 (SV40) promoter.

In particular embodiments, the 5′ LTR or 3′ LTR is a lentivirus LTR.

In particular embodiments, the 3′ LTR comprises one or moremodifications.

In some embodiments, the 3′ LTR comprises one or more deletions.

In certain embodiments, the 3′ LTR is a self-inactivating (SIN) LTR.

In some embodiments, the polyadenylation sequence is a bovine growthhormone polyadenylation or signal rabbit β-globin polyadenylationsequence.

In additional embodiments, a polynucleotide encoding a CAR contemplatedherein comprises an optimized Kozak sequence.

In further embodiments, the promoter operably linked to thepolynucleotide encoding a CAR contemplated herein is selected from thegroup consisting of: a cytomegalovirus immediate early gene promoter(CMV), an elongation factor 1 alpha promoter (EF1-α), a phosphoglyceratekinase-1 promoter (PGK), a ubiquitin-C promoter (UBQ-C), acytomegalovirus enhancer/chicken beta-actin promoter (CAG), polyomaenhancer/herpes simplex thymidine kinase promoter (MC1), a beta actinpromoter (β-ACT), a simian virus 40 promoter (SV40), and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer-binding site substituted (MND) promoter.

In various embodiments, an immune effector cell is provided comprising avector contemplated herein. In various embodiments, the immune effectorcell is transduced with a vector contemplated herein.

In further embodiments, the immune effector cell is selected from thegroup consisting of: a T lymphocyte and a natural killer (NK) cell.

In some embodiments, the immune effector cell is transduced with thevector of any one of the embodiments described above and is activatedand stimulated in the presence of an inhibitor of the PI3K pathway,thereby maintaining proliferation of the transduced immune effectorcells compared to the proliferation of transduced immune effector cellsthat were activated and stimulated in the absence of the inhibitor ofthe PI3K pathway.

In particular embodiments, the immune effector cell activated andstimulated in the presence of the inhibitor of PI3K pathway hasincreased expression of i) one or more markers selected from the groupconsisting of: CD62L, CD127, CD197, and CD38 or ii) all of the markersCD62L, CD127, CD197, and CD38 compared to an immune effector cellactivated and stimulated in the absence of the inhibitor of PI3Kpathway.

In one embodiment, the PI3K inhibitor is ZSTK474.

In various embodiments, a composition is provided comprising an immuneeffector cell contemplated herein and a physiologically acceptableexcipient.

In various embodiments, a method of generating an immune effector cellcomprising a CAR contemplated herein is provided, comprising introducinginto an immune effector cell a vector comprising a polynucleotideencoding the CAR.

In additional embodiments, the method further comprises stimulating theimmune effector cell and inducing the cell to proliferate by contactingthe cell with antibodies that bind CD3 and antibodies that bind to CD28;thereby generating a population of immune effector cells.

In particular embodiments, the immune effector cell is stimulated andinduced to proliferate before introducing the vector.

In certain embodiments, the immune effector cells comprise Tlymphocytes.

In particular embodiments, the immune effector cells comprise NK cells.

In particular embodiments, the cells are the activated and stimulated inthe presence of an inhibitor of the PI3K pathway, thereby maintainingproliferation of the transduced immune effector cells compared to theproliferation of immune effector cells that are activated and stimulatedin the absence of the inhibitor of the PI3K pathway

In some embodiments, the immune effector cells activated and stimulatedin the presence of the inhibitor of PI3K pathway have increasedexpression of i) one or more markers selected from the group consistingof: CD62L, CD127, CD197, and CD38 or ii) all of the markers CD62L,CD127, CD197, and CD38 compared to immune effector cells activated andstimulated in the absence of the inhibitor of PI3K pathway.

In one embodiment, the PI3K inhibitor is ZSTK474.

In various embodiments, a method of treating a B cell related conditionin a subject in need thereof is provided, comprising administering tothe subject a therapeutically effect amount of a composition comprisingBCMA CAR T cells contemplated herein and optionally, a pharmaceuticallyacceptable excipient.

In other embodiments, the B cell related condition is multiple myeloma,non-Hodgkin's lymphoma, B cell proliferations of uncertain malignantpotential, lymphomatoid granulomatosis, post-transplantlymphoproliferative disorder, an immunoregulatory disorder, rheumatoidarthritis, myasthenia gravis, idiopathic thrombocytopenia purpura,anti-phospholipid syndrome, Chagas' disease, Grave's disease, Wegener'sgranulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigusvulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome,ANCA associated vasculitis, Goodpasture's disease, Kawasaki disease,autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis,heavy-chain disease, primary or immunocyte-associated amyloidosis, ormonoclonal gammopathy of undetermined significance.

In further embodiments, the B cell related condition is a B cellmalignancy.

In certain embodiments, the B cell malignancy is multiple myeloma (MM)or non-Hodgkin's lymphoma (NHL).

In certain embodiments, the MM is selected from the group consisting of:overt multiple myeloma, smoldering multiple myeloma, plasma cellleukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma,solitary plasmacytoma of bone, and extramedullary plasmacytoma.

In some embodiments, the NHL is selected from the group consisting of:Burkitt lymphoma, chronic lymphocytic leukemia/small lymphocyticlymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,and mantle cell lymphoma.

In particular embodiments, the B cell related condition is a plasma cellmalignancy.

In further embodiments, the B cell related condition is an autoimmunedisease.

In additional embodiments, the autoimmune disease is systemic lupuserythematosus.

In certain embodiments, the B cell related condition is rheumatoidarthritis.

In particular embodiments, the B cell related condition is idiopathicthrombocytopenia purpura, or myasthenia gravis, or autoimmune hemolyticanemia.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic of murine B cell maturation antigen (muBCMA)CAR constructs.

FIG. 2a shows the amount of IFNg released from anti-BCMA02 CAR T cells,anti-BCMA10 CART cells, and CAR19ΔT cells after the cells wereco-cultured for 24 hours with K562 cells expressing BCMA.

FIG. 2b shows the amount of IFNg released from anti-BCMA02 CAR T cells,anti-BCMA10 CART cells, and CAR19Δ T cells after the cells wereco-cultured for 24 hours with K562 cells that lack BCMA expressioncompared to K562 cells expressing BCMA.

FIG. 3 shows the amount of inflammatory cytokines in growth media fromuntransduced control T cells, anti-BCMA02 CAR T cells, anti-BCMA10 CARTcells, and CAR19Δ T cells, stimulated 10 days prior to the assay.

FIG. 4 shows the amount of inflammatory cytokines produced byanti-BCMA02 CAR T cells, anti-BCMA10 CART cells, and CAR19Δ T cells inthe absence of antigen stimulation.

FIG. 5 shows the expression of phenotypic markers of activation at theend of anti-BCMA CAR T cell manufacturing. HLA-DR and CD25 expressionwas measured in anti-BCMA02 CAR T cells, anti-BCMA10 CART cells, andCAR19Δ T cells.

FIG. 6 shows the levels of activated caspase-3, a necessary step inapoptosis and important for AICD in anti-BCMA10 CAR T cells andanti-BCMA02 CART cells in the absence of antigen stimulation.

FIG. 7 shows the amount of inflammatory cytokine release in anti-BCMA02and anti-BCMA10 CART cells in media containing fetal bovine serum (FBS),human AB serum (HABS), or 100 ng/ml soluble BCMA.

FIG. 8A shows the tumor volume in NOD scid gamma (NSG) mice with ˜100mm³ experimental sub-cutaneous human multiple myeloma (RPMI-8226)tumors. Mice were treated with vehicle, 10⁷ anti-BCMA02 CAR T cells, 10⁷anti-BCMA10 CAR T cells, or Bortezomib (velcade).

FIG. 8B shows the tumor volume in NOD scid gamma (NSG) mice with ˜100mm³ experimental sub-cutaneous human multiple myeloma (RPMI-8226)tumors. Mice were treated with vehicle, 10⁷ anti-BCMA02 CAR T cells, 10⁷anti-BCMA10 CAR T cells, or Bortezomib (velcade).

FIG. 9 shows the level of BCMA expression on lymphoma and leukemia celllines (circles) and the activity of anti-BCMA CAR T cells to each cellline (IFNγ release, boxes). BCMA-negative (BCMA−) tumor cell lines:myelogenous leukemia (K562), acute lymphoblastic leukemia (NALM-6 andNALM-16); Mantle cell lymphoma (REC-1); or Hodgkin's lymphoma (HDLM-2)showed little or no IFNγ release. BCMA-positive (BCMA+) tumor celllines: B cell chronic lymphoblastic leukemia (MEC-1), Mantle celllymphoma (JeKo-1), Hodgkin's lymphoma (RPMI-6666), Burkitt's lymphoma(Daudi cells and Ramos cells), and multiple myeloma (RPMI-8226) showedsubstantial IFNγ release.

FIG. 10A shows the in vivo activity of vehicle, anti-CD19Δ CAR T cells,anti-CD19 CAR T cells, and anti-BCMA CAR T cells to BCMA expressingBurkitt's lymphoma cells (Daudi cells) in an NSG mouse model when CAR Tcells are administered to the mice at 8 days post tumor induction.

FIG. 10B shows the in vivo activity of vehicle, anti-CD19Δ CAR T cells,anti-CD19 CAR T cells, and anti-BCMA CAR T cells to BCMA expressingBurkitt's lymphoma cells (Daudi cells) in an NSG mouse model when CAR Tcells are administered to the mice at 18 days post tumor induction.

FIG. 11 shows potent in vitro activity of anti-BCMA CAR T cells achievedwith a 50 percent reduction anti-BCMA CAR expression. (A) T cellpopulations were transduced with between 4×10⁸ and 5×10⁷ transducingunits of a lentivirus encoding an anti-BCMA CAR molecule (MOI of 5 to40). The resulting T cell populations were normalized to contain 26±4%anti-BCMA CAR-positive T cells. (B) MFI of the normalized anti-BCMA CART cells ranged from 885 to 1875 as assayed by flow cytometry. (C) K562cells and K562-BCMA cells were co-cultured with normalized anti-BCMACART cells at a 20:1 or 10:1 effector (E; T cell) to target (T; 1:1 mixof K562 and K562 BCMA cells) ratio showed comparable cytolytic activity.

FIG. 12 shows the reliability of the manufacturing process for anti-BCMACAR T cells. (A) anti-BCMA CAR T cell products manufactured from PBMCsof 11 individual donors show comparable levels of expansion compared toa matched culture of untransduced donor T cells. (B) anti-BCMA CAR Tcell products manufactured from the 11 donors showed comparablelentiviral transduction efficiency (VCN). (C) The frequency of anti-BCMACAR positive T cells was measured by flow cytometry and BCMA expressionwas found to be comparable across all donors. (D) anti-BCMA CAR T cellproducts manufactured from the 11 donors showed therapeutically relevantlevels of IFNγ release when exposed to BCMA-expressing K562 cells.

FIG. 13 shows Venn diagrams for co-expression of CD127, CD197 and CD38in CD62L positive anti-BCMA02 T cells that have been cultured in thepresence of IL-2 or IL-2 and ZSTK474 for ten days. ZSTK474-treatedanti-BCMA02 CAR T cells showed an increase in the percentage of cellsco-expressing CD127, CD197 and CD38 compared to anti-BCMA CAR T cellscultured with IL-2 alone.

FIG. 14 shows an increased percentage of CD8 expressing anti-BCMA02 CART cells in cultures treated IL-2 and ZSTK474 (n=7) compared to culturestreated with IL-2 alone. CD8 expression was determined using afluorescently-labeled anti-CD8 antibody and flow cytometry.

FIG. 15 shows the amount of IFN-γ released by anti-BCMA02 CART cellsfrom 14 donors after culture with IL-2 alone or with IL-2 and ZSTK474.At the end of the culture period, an equivalent number of anti-BCMA02CAR T cells were re-cultured for 24 hours in media alone. The amount ofIFN-γ released in 24 hours was quantified by ELISA. Culture in ZSTK474did not significantly increase anti-BCMA02 CAR T cell tonic cytokinerelease compared to anti-BCMA02 CAR T cells cultured with IL-2 alone.

FIG. 16 shows anti-tumor activity of anti-BCMA02 CAR T cells treatedwith IL-2, or IL-2 and ZSTK474, or a truncated signaling deficientanti-BCMA02 (tBCMA02) CAR T cell treated with IL-2 and ZSTK474 in anaggressive Daudi tumor model. Complete tumor regression was observed in50% of mice administered the anti-BCMA02 CAR T cells treated with IL-2and ZSTK474.

FIG. 17 shows anti-tumor activity of anti-BCMA02 CAR T cells treatedwith IL-2, or IL-2 and ZSTK474 in a multiple myeloma tumor (RPMI-8226)model. Animals treated with IL-2- or IL-2 and ZSTK474-culturedanti-BCMA02 CAR T cells completely prevented tumor outgrowth.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NOs: 1-3 set forth amino acid sequences of exemplary light chainCDR sequences for BCMA CARs contemplated herein.

SEQ ID NOs: 4-6 set forth amino acid sequences of exemplary heavy chainCDR sequences for BCMA CARs contemplated herein.

SEQ ID NO: 7 sets forth an amino acid sequence of an exemplary lightchain sequences for BCMA CARs contemplated herein.

SEQ ID NO: 8 sets forth an amino acid sequence of an exemplary heavychain sequences for BCMA CARs contemplated herein.

SEQ ID NO: 9 sets forth an amino acid sequence of an exemplary BCMA CARcontemplated herein.

SEQ ID NO: 10 set forth a polynucleotide sequence that encode anexemplary BCMA CAR contemplated herein.

SEQ ID NO: 11 sets forth the amino acid sequence of human BCMA.

SEQ ID NO: 12-22 set for the amino acid sequence of various linkers.

SEQ ID NOs: 23-35 set for the amino acid sequence of protease cleavagesites and self-cleaving polypeptide cleavage sites.

SEQ ID NO: 36 sets for the polynucleotide sequence of a vector encodinga BCMA CAR.

DETAILED DESCRIPTION

A. Overview

The invention generally relates to improved compositions and methods fortreating B cell related conditions. As used herein, the term “B cellrelated conditions” relates to conditions involving inappropriate B cellactivity and B cell malignancies.

In particular embodiments, the invention relates to improved adoptivecell therapy of B cell related conditions using genetically modifiedimmune effector cells. Genetic approaches offer a potential means toenhance immune recognition and elimination of cancer cells. Onepromising strategy is to genetically engineer immune effector cells toexpress chimeric antigen receptors (CAR) that redirect cytotoxicitytoward cancer cells. However, existing adoptive cell immunotherapies fortreating B cell disorders present a serious risk of compromising humoralimmunity because the cells target antigens expressed on all of, or themajority of, B cells. Accordingly, such therapies are not clinicallydesirable and thus, a need in the art remains for more efficienttherapies for B cell related conditions that spare humoral immunity.

The improved compositions and methods of adoptive cell therapy disclosedherein, provide genetically modified immune effector cells that canreadily be expanded, exhibit long-term persistence in vivo, and reduceimpairment of humoral immunity by targeting B cells expression B cellmaturation antigen (BCMA, also known as CD269 or tumor necrosis factorreceptor superfamily, member 17; TNFRSF17).

BCMA is a member of the tumor necrosis factor receptor superfamily (see,e.g., Thompson et al., J. Exp. Medicine, 192(1): 129-135, 2000, andMackay et al., Annu. Rev. Immunol, 21: 231-264, 2003. BCMA binds B-cellactivating factor (BAFF) and a proliferation inducing ligand (APRIL)(see, e.g., Mackay et al., 2003 and Kalled et al., ImmunologicalReviews, 204: 43-54, 2005). Among nonmalignant cells, BCMA has beenreported to be expressed mostly in plasma cells and subsets of matureB-cells (see, e.g., Laabi et al., EMBO J., 77(1): 3897-3904, 1992; Laabiet al., Nucleic Acids Res., 22(7): 1147-1154, 1994; Kalled et al., 2005;O'Connor et al., J. Exp. Medicine, 199(1): 91-97, 2004; and Ng et al.,J. Immunol., 73(2): 807-817, 2004. Mice deficient in BCMA are healthyand have normal numbers of B cells, but the survival of long-livedplasma cells is impaired (see, e.g., O'Connor et al., 2004; Xu et al.,Mol. Cell. Biol, 21(12): 4067-4074, 2001; and Schiemann et al., Science,293(5537): 2 111-21 14, 2001). BCMA RNA has been detected universally inmultiple myeloma cells and in other lymphomas, and BCMA protein has beendetected on the surface of plasma cells from multiple myeloma patientsby several investigators (see, e.g., Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19): 5903-5909,2007; Bellucci et al., Blood, 105(10): 3945-3950, 2005; and Moreaux etal., Blood, 703(8): 3148-3157, 2004.

In various embodiments, CARs comprising murine anti-BCMA antibodysequences are highly efficacious compared to BCMA CARs comprisingparticular human antibody sequences; undergo robust in vivo expansion;and recognize human B cells expressing BMCA; show cytotoxic activityagainst the BCMA expressing B cells; and do not show signs of inducing acytokine storm, a potentially fatal condition where the cytokinesreleased by activated T cells create a sudden inflammatory response inthe system that spurs a noninfectious fever.

In one embodiment, a CAR comprising a murine anti-BCMA antibody orantigen binding fragment, a transmembrane domain, and one or moreintracellular signaling domains is provided.

In one embodiment, an immune effector cell is genetically modified toexpress a CAR contemplated herein is provided. T cells expressing a CARare referred to herein as CART cells or CAR modified T cells.

In various embodiments, the genetically modified immune effector cellscontemplated herein, are administered to a patient with a B cell relatedcondition, e.g., an autoimmune disease associated with B cells or a Bcell malignancy.

The practice of the invention will employ, unless indicated specificallyto the contrary, conventional methods of chemistry, biochemistry,organic chemistry, molecular biology, microbiology, recombinant DNAtechniques, genetics, immunology, and cell biology that are within theskill of the art, many of which are described below for the purpose ofillustration. Such techniques are explained fully in the literature.See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rdEdition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual (1982); Ausubel et al., Current Protocols in Molecular Biology(John Wiley and Sons, updated July 2008); Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates and Wiley-Interscience; Glover, DNACloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985);Anand, Techniques for the Analysis of Complex Genomes, (Academic Press,New York, 1992); Transcription and Translation (B. Hames & S. Higgins,Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984);Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober,eds., 1991); Annual Review of Immunology; as well as monographs injournals such as Advances in Immunology.

B. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred embodimentsof compositions, methods and materials are described herein. For thepurposes of the present invention, the following terms are definedbelow.

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one, or to one or more) of thegrammatical object of the article. By way of example, “an element” meansone element or one or more elements.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “and/or” should be understood to mean either one, or both ofthe alternatives.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are present that materially affect the activity or action ofthe listed elements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments. It is also understood that the positive recitation of afeature in one embodiment, serves as a basis for excluding the featurein a particular embodiment.

C. Chimeric Antigen Receptors

In various embodiments, genetically engineered receptors that redirectcytotoxicity of immune effector cells toward B cells are provided. Thesegenetically engineered receptors referred to herein as chimeric antigenreceptors (CARs). CARs are molecules that combine antibody-basedspecificity for a desired antigen (e.g., BCMA) with a T cellreceptor-activating intracellular domain to generate a chimeric proteinthat exhibits a specific anti-BCMA cellular immune activity. As usedherein, the term, “chimeric,” describes being composed of parts ofdifferent proteins or DNAs from different origins.

CARs contemplated herein, comprise an extracellular domain (alsoreferred to as a binding domain or antigen-specific binding domain) thatbinds to BCMA, a transmembrane domain, and an intracellular signalingdomain. Engagement of the anti-BCMA antigen binding domain of the CARwith BCMA on the surface of a target cell results in clustering of theCAR and delivers an activation stimulus to the CAR-containing cell. Themain characteristic of CARs are their ability to redirect immuneeffector cell specificity, thereby triggering proliferation, cytokineproduction, phagocytosis or production of molecules that can mediatecell death of the target antigen expressing cell in a majorhistocompatibility (MHC) independent manner, exploiting the cellspecific targeting abilities of monoclonal antibodies, soluble ligandsor cell specific co-receptors.

In various embodiments, a CAR comprises an extracellular binding domainthat comprises a murine anti-BCMA-specific binding domain; atransmembrane domain; one or more intracellular co-stimulatory signalingdomains; and a primary signaling domain.

In particular embodiments, a CAR comprises an extracellular bindingdomain that comprises a murine anti-BCMA antibody or antigen bindingfragment thereof; one or more hinge domains or spacer domains; atransmembrane domain including; one or more intracellular co-stimulatorysignaling domains; and a primary signaling domain.

1. Binding Domain

In particular embodiments, CARs contemplated herein comprise anextracellular binding domain that comprises a murine anti-BCMA antibodyor antigen binding fragment thereof that specifically binds to a humanBCMA polypeptide expressed on a B cell. As used herein, the terms,“binding domain,” “extracellular domain,” “extracellular bindingdomain,” “antigen-specific binding domain,” and “extracellular antigenspecific binding domain,” are used interchangeably and provide a CARwith the ability to specifically bind to the target antigen of interest,e.g., BCMA. The binding domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source.

The terms “specific binding affinity” or “specifically binds” or“specifically bound” or “specific binding” or “specifically targets” asused herein, describe binding of an anti-BCMA antibody or antigenbinding fragment thereof (or a CAR comprising the same) to BCMA atgreater binding affinity than background binding. A binding domain (or aCAR comprising a binding domain or a fusion protein containing a bindingdomain) “specifically binds” to a BCMA if it binds to or associates withBCMA with an affinity or K_(a) (i.e., an equilibrium associationconstant of a particular binding interaction with units of 1/M) of, forexample, greater than or equal to about 10⁵ M⁻¹. In certain embodiments,a binding domain (or a fusion protein thereof) binds to a target with aKa greater than or equal to about 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹,10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹. “High affinity” bindingdomains (or single chain fusion proteins thereof) refers to thosebinding domains with a K_(a) of at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, atleast 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 10¹² M⁻¹,at least 10¹³ M⁻¹, or greater.

Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(d)) of a particular binding interaction with units of M(e.g., 10⁻⁵ M to 10⁻¹³ M, or less). Affinities of binding domainpolypeptides and CAR proteins according to the present disclosure can bereadily determined using conventional techniques, e.g., by competitiveELISA (enzyme-linked immunosorbent assay), or by binding association, ordisplacement assays using labeled ligands, or using a surface-plasmonresonance device such as the Biacore T100, which is available fromBiacore, Inc., Piscataway, N.J., or optical biosensor technology such asthe EPIC system or EnSpire that are available from Corning and PerkinElmer respectively (see also, e.g., Scatchard et al. (1949) Ann. N.Y.Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or theequivalent).

In one embodiment, the affinity of specific binding is about 2 timesgreater than background binding, about 5 times greater than backgroundbinding, about 10 times greater than background binding, about 20 timesgreater than background binding, about 50 times greater than backgroundbinding, about 100 times greater than background binding, or about 1000times greater than background binding or more.

In particular embodiments, the extracellular binding domain of a CARcomprises an antibody or antigen binding fragment thereof. An “antibody”refers to a binding agent that is a polypeptide comprising at least alight chain or heavy chain immunoglobulin variable region whichspecifically recognizes and binds an epitope of an antigen, such as apeptide, lipid, polysaccharide, or nucleic acid containing an antigenicdeterminant, such as those recognized by an immune cell.

An “antigen (Ag)” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T cell response in ananimal, including compositions (such as one that includes acancer-specific protein) that are injected or absorbed into an animal.An antigen reacts with the products of specific humoral or cellularimmunity, including those induced by heterologous antigens, such as thedisclosed antigens. In particular embodiments, the target antigen is anepitope of a BCMA polypeptide.

An “epitope” or “antigenic determinant” refers to the region of anantigen to which a binding agent binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or about 8-10 amino acids in a uniquespatial conformation.

Antibodies include antigen binding fragments thereof, such as Camel Ig,Ig NAR, Fab fragments, Fab′ fragments, F(ab)′2 fragments, F(ab)′3fragments, Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)₂,minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fvproteins (“dsFv”), and single-domain antibody (sdAb, Nanobody) andportions of full length antibodies responsible for antigen binding. Theterm also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies) and antigen bindingfragments thereof. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3_(rd) Ed.,W. H. Freeman & Co., New York, 1997.

As would be understood by the skilled person and as described elsewhereherein, a complete antibody comprises two heavy chains and two lightchains. Each heavy chain consists of a variable region and a first,second, and third constant region, while each light chain consists of avariable region and a constant region. Mammalian heavy chains areclassified as α, δ, ε, γ, and μ. Mammalian light chains are classifiedas λ or κ. Immunoglobulins comprising the α, δ, ε, γ, and μ heavy chainsare classified as immunoglobulin (Ig)A, IgD, IgE, IgG, and IgM. Thecomplete antibody forms a “Y” shape. The stem of the Y consists of thesecond and third constant regions (and for IgE and IgM, the fourthconstant region) of two heavy chains bound together and disulfide bonds(inter-chain) are formed in the hinge. Heavy chains γ, α and δ have aconstant region composed of three tandem (in a line) Ig domains, and ahinge region for added flexibility; heavy chains μ and ε have a constantregion composed of four immunoglobulin domains. The second and thirdconstant regions are referred to as “CH2 domain” and “CH3 domain”,respectively. Each arm of the Y includes the variable region and firstconstant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding.

Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The CDRs can be definedor identified by conventional methods, such as by sequence according toKabat et al (Wu, T T and Kabat, E. A., J Exp Med. 132(2):211-50, (1970);Borden, P. and Kabat E. A., PNAS, 84: 2440-2443 (1987); (see, Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference), or by structure according to Chothia et al (Choithia, C. andLesk, A. M., J Mol. Biol., 196(4): 901-917 (1987), Choithia, C. et al,Nature, 342: 877-883 (1989)).

The sequences of the framework regions of different light or heavychains are relatively conserved within a species, such as humans. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three-dimensional space. The CDRs are primarily responsiblefor binding to an epitope of an antigen. The CDRs of each chain aretypically referred to as CDR1, CDR2, and CDR3, numbered sequentiallystarting from the N-terminus, and are also typically identified by thechain in which the particular CDR is located. Thus, the CDRs located inthe variable domain of the heavy chain of the antibody are referred toas CDRH1, CDRH2, and CDRH3, whereas the CDRs located in the variabledomain of the light chain of the antibody are referred to as CDRL1,CDRL2, and CDRL3. Antibodies with different specificities (i.e.,different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs). Illustrative examples of lightchain CDRs that are suitable for constructing humanized BCMA CARscontemplated herein include, but are not limited to the CDR sequencesset forth in SEQ ID NOs: 1-3. Illustrative examples of heavy chain CDRsthat are suitable for constructing humanized BCMA CARs contemplatedherein include, but are not limited to the CDR sequences set forth inSEQ ID NOs: 4-6.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an antibody, Fv, scFv,dsFv, Fab, or other antibody fragment as disclosed herein. References to“V_(L)” or “VL” refer to the variable region of an immunoglobulin lightchain, including that of an antibody, Fv, scFv, dsFv, Fab, or otherantibody fragment as disclosed herein.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a mouse. In particular preferred embodiments, a CARcontemplated herein comprises antigen-specific binding domain that is achimeric antibody or antigen binding fragment thereof.

A “humanized” antibody is an immunoglobulin including a human frameworkregion and one or more CDRs from a non-human (for example a mouse, rat,or synthetic) immunoglobulin. The non-human immunoglobulin providing theCDRs is termed a “donor,” and the human immunoglobulin providing theframework is termed an “acceptor.”

In particular embodiments, a murine anti-BCMA antibody or antigenbinding fragment thereof, includes but is not limited to a Camel Ig (acamelid antibody (VHH)), Ig NAR, Fab fragments, Fab′ fragments, F(ab)′2fragments, F(ab)′3 fragments, Fv, single chain Fv antibody (“scFv”),bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfidestabilized Fv protein (“dsFv”), and single-domain antibody (sdAb,Nanobody).

“Camel Ig” or “camelid VHH” as used herein refers to the smallest knownantigen-binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEBJ., 21: 3490-3498 (2007)). A “heavy chain antibody” or a “camelidantibody” refers to an antibody that contains two VH domains and nolight chains (Riechmann L. et al, J. Immunol. Methods 231:25-38 (1999);WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079).

“IgNAR” of “immunoglobulin new antigen receptor” refers to class ofantibodies from the shark immune repertoire that consist of homodimersof one variable new antigen receptor (VNAR) domain and five constant newantigen receptor (CNAR) domains. IgNARs represent some of the smallestknown immunoglobulin-based protein scaffolds and are highly stable andpossess efficient binding characteristics. The inherent stability can beattributed to both (i) the underlying Ig scaffold, which presents aconsiderable number of charged and hydrophilic surface exposed residuescompared to the conventional antibody VH and VL domains found in murineantibodies; and (ii) stabilizing structural features in thecomplementary determining region (CDR) loops including inter-loopdisulphide bridges, and patterns of intra-loop hydrogen bonds.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three hypervariableregions (HVRs) of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six HVRs confer antigen-binding specificity to the antibody.However, even a single variable domain (or half of an Fv comprising onlythree HVRs specific for an antigen) has the ability to recognize andbind antigen, although at a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., PNAS USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

“Single domain antibody” or “sdAb” or “nanobody” refers to an antibodyfragment that consists of the variable region of an antibody heavy chain(VH domain) or the variable region of an antibody light chain (VLdomain) (Holt, L., et al, Trends in Biotechnology, 21(11): 484-490).

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain and in either orientation (e.g., VL-VH or VH-VL).Generally, the scFv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains which enables the scFv to form the desiredstructure for antigen binding. For a review of scFv, see, e.g.,Pluckthiin, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

In preferred embodiments, a CAR contemplated herein comprisesantigen-specific binding domain that is a murine scFv. Single chainantibodies may be cloned form the V region genes of a hybridoma specificfor a desired target. The production of such hybridomas has becomeroutine. A technique which can be used for cloning the variable regionheavy chain (V_(H)) and variable region light chain (V_(L)) has beendescribed, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.

In particular embodiments, the antigen-specific binding domain that is amurine scFv that binds a human BCMA polypeptide. Illustrative examplesof variable heavy chains that are suitable for constructing BCMA CARscontemplated herein include, but are not limited to the amino acidsequences set forth in SEQ ID NO: 8. Illustrative examples of variablelight chains that are suitable for constructing BCMA CARs contemplatedherein include, but are not limited to the amino acid sequences setforth in SEQ ID NO: 7.

BCMA-specific binding domains provided herein also comprise one, two,three, four, five, or six CDRs. Such CDRs may be nonhuman CDRs oraltered nonhuman CDRs selected from CDRL1, CDRL2 and CDRL3 of the lightchain and CDRH1, CDRH2 and CDRH3 of the heavy chain. In certainembodiments, a BCMA-specific binding domain comprises (a) a light chainvariable region that comprises a light chain CDRL1, a light chain CDRL2,and a light chain CDRL3, and (b) a heavy chain variable region thatcomprises a heavy chain CDRH1, a heavy chain CDRH2, and a heavy chainCDRH3.

2. Linkers

In certain embodiments, the CARs contemplated herein may comprise linkerresidues between the various domains, e.g., added for appropriatespacing and conformation of the molecule. In particular embodiments thelinker is a variable region linking sequence. A “variable region linkingsequence,” is an amino acid sequence that connects the V_(H) and V_(L)domains and provides a spacer function compatible with interaction ofthe two sub-binding domains so that the resulting polypeptide retains aspecific binding affinity to the same target molecule as an antibodythat comprises the same light and heavy chain variable regions. CARscontemplated herein, may comprise one, two, three, four, or five or morelinkers. In particular embodiments, the length of a linker is about 1 toabout 25 amino acids, about 5 to about 20 amino acids, or about 10 toabout 20 amino acids, or any intervening length of amino acids. In someembodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acidslong.

Illustrative examples of linkers include glycine polymers (G)_(n);glycine-serine polymers (G₁₋₅S₁₋₅)_(n), where n is an integer of atleast one, two, three, four, or five; glycine-alanine polymers;alanine-serine polymers; and other flexible linkers known in the art.Glycine and glycine-serine polymers are relatively unstructured, andtherefore may be able to serve as a neutral tether between domains offusion proteins such as the CARs described herein. Glycine accessessignificantly more phi-psi space than even alanine, and is much lessrestricted than residues with longer side chains (see Scheraga, Rev.Computational Chem. 11173-142 (1992)). The ordinarily skilled artisanwill recognize that design of a CAR in particular embodiments caninclude linkers that are all or partially flexible, such that the linkercan include a flexible linker as well as one or more portions thatconfer less flexible structure to provide for a desired CAR structure.

Other exemplary linkers include, but are not limited to the followingamino acid sequences: GGG; DGGGS (SEQ ID NO: 12); TGEKP (SEQ ID NO: 13)(see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 14)(Pomerantz et al. 1995, supra); (GGGGS)_(n) wherein=1, 2, 3, 4 or 5 (SEQID NO: 15) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQID NO: 16) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 17) (Bird et al., 1988,Science 242:423-426), GGRRGGGS (SEQ ID NO: 18); LRQRDGERP (SEQ ID NO:19); LRQKDGGGSERP (SEQ ID NO: 20); LRQKd(GGGS)₂ ERP (SEQ ID NO: 21).Alternatively, flexible linkers can be rationally designed using acomputer program capable of modeling both DNA-binding sites and thepeptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS91:11099-11103 (1994) or by phage display methods. In one embodiment,the linker comprises the following amino acid sequence:GSTSGSGKPGSGEGSTKG (SEQ ID NO: 22) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

3. Spacer Domain

In particular embodiments, the binding domain of the CAR is followed byone or more “spacer domains,” which refers to the region that moves theantigen binding domain away from the effector cell surface to enableproper cell/cell contact, antigen binding and activation (Patel et al.,Gene Therapy, 1999; 6: 412-419). The hinge domain may be derived eitherfrom a natural, synthetic, semi-synthetic, or recombinant source. Incertain embodiments, a spacer domain is a portion of an immunoglobulin,including, but not limited to, one or more heavy chain constant regions,e.g., CH2 and CH3. The spacer domain can include the amino acid sequenceof a naturally occurring immunoglobulin hinge region or an alteredimmunoglobulin hinge region.

In one embodiment, the spacer domain comprises the CH2 and CH3 domainsof IgG1 or IgG4.

4. Hinge Domain

The binding domain of the CAR is generally followed by one or more“hinge domains,” which plays a role in positioning the antigen bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen binding and activation. A CAR generally comprises oneor more hinge domains between the binding domain and the transmembranedomain (TM). The hinge domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source. The hinge domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region.

An “altered hinge region” refers to (a) a naturally occurring hingeregion with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%,10%, or 5% amino acid substitutions or deletions), (b) a portion of anaturally occurring hinge region that is at least 10 amino acids (e.g.,at least 12, 13, 14 or 15 amino acids) in length with up to 30% aminoacid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acidsubstitutions or deletions), or (c) a portion of a naturally occurringhinge region that comprises the core hinge region (which may be 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 amino acids in length). In certain embodiments,one or more cysteine residues in a naturally occurring immunoglobulinhinge region may be substituted by one or more other amino acid residues(e.g., one or more serine residues). An altered immunoglobulin hingeregion may alternatively or additionally have a proline residue of awild type immunoglobulin hinge region substituted by another amino acidresidue (e.g., a serine residue).

Other illustrative hinge domains suitable for use in the CARs describedherein include the hinge region derived from the extracellular regionsof type 1 membrane proteins such as CD8α, CD4, CD28 and CD7, which maybe wild-type hinge regions from these molecules or may be altered. Inanother embodiment, the hinge domain comprises a CD8α hinge region.

5. Transmembrane (TM) Domain

The “transmembrane domain” is the portion of the CAR that fuses theextracellular binding portion and intracellular signaling domain andanchors the CAR to the plasma membrane of the immune effector cell. TheTM domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The TM domain may be derived from(i.e., comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9,CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134,CD137, CD152, CD 154, and PD1. In a particular embodiment, the TM domainis synthetic and predominantly comprises hydrophobic residues such asleucine and valine.

In one embodiment, the CARs contemplated herein comprise a TM domainderived from CD8α. In another embodiment, a CAR contemplated hereincomprises a TM domain derived from CD8α and a short oligo- orpolypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids in length that links the TM domain and the intracellularsignaling domain of the CAR. A glycine-serine based linker provides aparticularly suitable linker.

6. Intracellular Signaling Domain

In particular embodiments, CARs contemplated herein comprise anintracellular signaling domain. An “intracellular signaling domain,”refers to the part of a CAR that participates in transducing the messageof effective BCMA CAR binding to a human BCMA polypeptide into theinterior of the immune effector cell to elicit effector cell function,e.g., activation, cytokine production, proliferation and cytotoxicactivity, including the release of cytotoxic factors to the CAR-boundtarget cell, or other cellular responses elicited with antigen bindingto the extracellular CAR domain.

The term “effector function” refers to a specialized function of animmune effector cell. Effector function of the T cell, for example, maybe cytolytic activity or help or activity including the secretion of acytokine. Thus, the term “intracellular signaling domain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces the effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingdomains: primary signaling domains that initiate antigen-dependentprimary activation through the TCR (e.g., a TCR/CD3 complex) andco-stimulatory signaling domains that act in an antigen-independentmanner to provide a secondary or co-stimulatory signal. In preferredembodiments, a CAR contemplated herein comprises an intracellularsignaling domain that comprises one or more “co-stimulatory signalingdomain” and a “primary signaling domain.”

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs.

Illustrative examples of ITAM containing primary signaling domains thatare of particular use in the invention include those derived from TCRζ,FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Inparticular preferred embodiments, a CAR comprises a CD3ζ primarysignaling domain and one or more co-stimulatory signaling domains. Theintracellular primary signaling and co-stimulatory signaling domains maybe linked in any order in tandem to the carboxyl terminus of thetransmembrane domain.

CARs contemplated herein comprise one or more co-stimulatory signalingdomains to enhance the efficacy and expansion of T cells expressing CARreceptors. As used herein, the term, “co-stimulatory signaling domain,”or “co-stimulatory domain”, refers to an intracellular signaling domainof a co-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, a CAR comprises one or more co-stimulatory signaling domainsselected from the group consisting of CD28, CD137, and CD134, and a CD3ζprimary signaling domain.

In another embodiment, a CAR comprises CD28 and CD137 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In yet another embodiment, a CAR comprises CD28 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In one embodiment, a CAR comprises CD137 and CD134 co-stimulatorysignaling domains and a CD3ζ primary signaling domain.

In particular embodiments, CARs contemplated herein comprise a murineanti-BCMA antibody or antigen binding fragment thereof that specificallybinds to a BCMA polypeptide expressed on B cells.

In one embodiment, a CAR comprises a murine anti-BCMA scFv that binds aBCMA polypeptide; a transmembrane domain derived from a polypeptideselected from the group consisting of: alpha, beta or zeta chain of theT-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27,CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154,and PD1; and one or more intracellular co-stimulatory signaling domainsfrom a co-stimulatory molecule selected from the group consisting of:CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134(OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3),CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT,NKD2C SLP76, TRIM, and ZAP70; and a primary signaling domain from TCRζ,FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In one embodiment, a CAR comprises a murine anti-BCMA scFv that binds aBCMA polypeptide; a hinge domain selected from the group consisting of:IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, and a CD8α hinge; atransmembrane domain derived from a polypeptide selected from the groupconsisting of: alpha, beta or zeta chain of the T-cell receptor, CD3ε,CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45,CD64, CD80, CD86, CD 134, CD137, CD152, CD 154, and PD1; and one or moreintracellular co-stimulatory signaling domains from a co-stimulatorymolecule selected from the group consisting of: CARD11, CD2, CD7, CD27,CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150(SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2),CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70;and a primary signaling domain from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD38,CD3ζ, CD22, CD79a, CD79b, and CD66d.

In one embodiment, a CAR comprises a murine anti-BCMA scFv that binds aBCMA polypeptide; a hinge domain selected from the group consisting of:IgG1 hinge/CH2/CH3, IgG4 hinge/CH2/CH3, and a CD8α hinge; atransmembrane domain derived from a polypeptide selected from the groupconsisting of: alpha, beta or zeta chain of the T-cell receptor, CD3ε,CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45,CD64, CD80, CD86, CD 134, CD137, CD152, CD 154, and PD1; a short oligo-or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids in length that links the TM domain to the intracellularsignaling domain of the CAR; and one or more intracellularco-stimulatory signaling domains from a co-stimulatory molecule selectedfrom the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40,CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152(CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278(ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and a primarysignaling domain from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22,CD79a, CD79b, and CD66d.

In a particular embodiment, a CAR comprises a murine anti-BCMA scFv thatbinds a BCMA polypeptide; a hinge domain comprising an IgG1hinge/CH2/CH3 polypeptide and a CD8α polypeptide; a CD8α transmembranedomain comprising a polypeptide linker of about 3 to about 10 aminoacids; a CD137 intracellular co-stimulatory signaling domain; and a CD3ζprimary signaling domain.

In a particular embodiment, a CAR comprises a murine anti-BCMA scFv thatbinds a BCMA polypeptide; a hinge domain comprising a CD8α polypeptide;a CD8a transmembrane domain comprising a polypeptide linker of about 3to about 10 amino acids; a CD134 intracellular co-stimulatory signalingdomain; and a CD3ζ primary signaling domain.

In a particular embodiment, a CAR comprises a murine anti-BCMA scFv thatbinds a BCMA polypeptide; a hinge domain comprising a CD8α polypeptide;a CD8α transmembrane domain comprising a polypeptide linker of about 3to about 10 amino acids; a CD28 intracellular co-stimulatory signalingdomain; and a CD3ζ primary signaling domain.

Moreover, the design of the CARs contemplated herein enable improvedexpansion, long-term persistence, and tolerable cytotoxic properties inT cells expressing the CARs compared to non-modified T cells or T cellsmodified to express other CARs.

D. Polypeptides

The present invention contemplates, in part, CAR polypeptides andfragments thereof, cells and compositions comprising the same, andvectors that express polypeptides. In preferred embodiments, apolypeptide comprising one or more CARs as set forth in SEQ ID NO: 9 isprovided.

“Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are usedinterchangeably, unless specified to the contrary, and according toconventional meaning, i.e., as a sequence of amino acids. Polypeptidesare not limited to a specific length, e.g., they may comprise a fulllength protein sequence or a fragment of a full length protein, and mayinclude post-translational modifications of the polypeptide, forexample, glycosylations, acetylations, phosphorylations and the like, aswell as other modifications known in the art, both naturally occurringand non-naturally occurring. In various embodiments, the CARpolypeptides contemplated herein comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. Illustrativeexamples of suitable signal sequences useful in CARs disclosed hereininclude, but are not limited to the IgG1 heavy chain signal sequence andthe CD8α signal sequence. Polypeptides can be prepared using any of avariety of well-known recombinant and/or synthetic techniques.Polypeptides contemplated herein specifically encompass the CARs of thepresent disclosure, or sequences that have deletions from, additions to,and/or substitutions of one or more amino acid of a CAR as disclosedherein.

An “isolated peptide” or an “isolated polypeptide” and the like, as usedherein, refer to in vitro isolation and/or purification of a peptide orpolypeptide molecule from a cellular environment, and from associationwith other components of the cell, i.e., it is not significantlyassociated with in vivo substances. Similarly, an “isolated cell” refersto a cell that has been obtained from an in vivo tissue or organ and issubstantially free of extracellular matrix.

Polypeptides include “polypeptide variants.” Polypeptide variants maydiffer from a naturally occurring polypeptide in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying one or more of the above polypeptide sequences. Forexample, in particular embodiments, it may be desirable to improve thebinding affinity and/or other biological properties of the CARs byintroducing one or more substitutions, deletions, additions and/orinsertions into a binding domain, hinge, TM domain, co-stimulatorysignaling domain or primary signaling domain of a CAR polypeptide.Preferably, polypeptides of the invention include polypeptides having atleast about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acididentity thereto.

Polypeptides include “polypeptide fragments.” Polypeptide fragmentsrefer to a polypeptide, which can be monomeric or multimeric, that hasan amino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion or substitution of a naturally-occurring orrecombinantly-produced polypeptide. In certain embodiments, apolypeptide fragment can comprise an amino acid chain at least 5 toabout 500 amino acids long. It will be appreciated that in certainembodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350,400, or 450 amino acids long. Particularly useful polypeptide fragmentsinclude functional domains, including antigen-binding domains orfragments of antibodies. In the case of a murine anti-BCMA antibody,useful fragments include, but are not limited to: a CDR region, a CDR3region of the heavy or light chain; a variable region of a heavy orlight chain; a portion of an antibody chain or variable region includingtwo CDRs; and the like.

The polypeptide may also be fused in-frame or conjugated to a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support.

As noted above, polypeptides of the invention may be altered in variousways including amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of a referencepolypeptide can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S.Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of theGene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.).

In certain embodiments, a variant will contain conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Modifications may be made in the structure ofthe polynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a variant or derivativepolypeptide with desirable characteristics. When it is desired to alterthe amino acid sequence of a polypeptide to create an equivalent, oreven an improved, variant polypeptide of the invention, one skilled inthe art, for example, can change one or more of the codons of theencoding DNA sequence, e.g., according to Table 1.

TABLE 1 Amino Acid Codons One Three letter letter Amino Acids code codeCodons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGUAspartic acid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine FPhe UUC UUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAUIsoleucine I Iso AUA AUC AUU Lysine K Lys AAA AAG Leucine L LeuUUA UUG CUA CUC CUG CUU Methionine M Met AUG Asparagine N Asn AAC AAUProline P Pro CCA CCC CCG CCU Glutamine Q Gln CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGU Serine S Ser AGC AGU UCA UCC UCG UCU Threonine TThr ACA ACC ACG ACU Valine V Val GUA GUC GUG GUU Tryptophan W Trp UGGTyrosine Y Tyr UAC UAU

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs well known in the art, such as DNASTAR™software. Preferably, amino acid changes in the protein variantsdisclosed herein are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In a peptide or protein, suitable conservativesubstitutions of amino acids are known to those of skill in this art andgenerally can be made without altering a biological activity of aresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., p. 224). Exemplary conservativesubstitutions are described in U.S. Provisional Patent Application No.61/241,647, the disclosure of which is herein incorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics (Kyte andDoolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions may be based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like.

Polypeptide variants further include glycosylated forms, aggregativeconjugates with other molecules, and covalent conjugates with unrelatedchemical moieties (e.g., pegylated molecules). Covalent variants can beprepared by linking functionalities to groups which are found in theamino acid chain or at the N- or C-terminal residue, as is known in theart. Variants also include allelic variants, species variants, andmuteins. Truncations or deletions of regions which do not affectfunctional activity of the proteins are also variants.

In one embodiment, where expression of two or more polypeptides isdesired, the polynucleotide sequences encoding them can be separated byand IRES sequence as discussed elsewhere herein. In another embodiment,two or more polypeptides can be expressed as a fusion protein thatcomprises one or more self-cleaving polypeptide sequences.

Polypeptides of the present invention include fusion polypeptides. Inpreferred embodiments, fusion polypeptides and polynucleotides encodingfusion polypeptides are provided, e.g., CARs. Fusion polypeptides andfusion proteins refer to a polypeptide having at least two, three, four,five, six, seven, eight, nine, or ten or more polypeptide segments.Fusion polypeptides are typically linked C-terminus to N-terminus,although they can also be linked C-terminus to C-terminus, N-terminus toN-terminus, or N-terminus to C-terminus. The polypeptides of the fusionprotein can be in any order or a specified order. Fusion polypeptides orfusion proteins can also include conservatively modified variants,polymorphic variants, alleles, mutants, subsequences, and interspecieshomologs, so long as the desired transcriptional activity of the fusionpolypeptide is preserved. Fusion polypeptides may be produced bychemical synthetic methods or by chemical linkage between the twomoieties or may generally be prepared using other standard techniques.Ligated DNA sequences comprising the fusion polypeptide are operablylinked to suitable transcriptional or translational control elements asdiscussed elsewhere herein.

In one embodiment, a fusion partner comprises a sequence that assists inexpressing the protein (an expression enhancer) at higher yields thanthe native recombinant protein. Other fusion partners may be selected soas to increase the solubility of the protein or to enable the protein tobe targeted to desired intracellular compartments or to facilitatetransport of the fusion protein through the cell membrane.

Fusion polypeptides may further comprise a polypeptide cleavage signalbetween each of the polypeptide domains described herein. In addition,polypeptide site can be put into any linker peptide sequence. Exemplarypolypeptide cleavage signals include polypeptide cleavage recognitionsites such as protease cleavage sites, nuclease cleavage sites (e.g.,rare restriction enzyme recognition sites, self-cleaving ribozymerecognition sites), and self-cleaving viral oligopeptides (see deFelipeand Ryan, 2004. Traffic, 5(8); 616-26).

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., in Ryan et al., 1997. J. Gener. Virol.78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus NIa proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.Due to its high cleavage stringency, TEV (tobacco etch virus) proteasecleavage sites are preferred in one embodiment, e.g., EXXYXQ(G/S) (SEQID NO: 23), for example, ENLYFQG (SEQ ID NO: 24) and ENLYFQS (SEQ ID NO:25), wherein X represents any amino acid (cleavage by TEV occurs betweenQ and G or Q and S).

In a particular embodiment, self-cleaving peptides include thosepolypeptide sequences obtained from potyvirus and cardiovirus 2Apeptides, FMDV (foot-and-mouth disease virus), equine rhinitis A virus,Thosea asigna virus and porcine teschovirus.

In certain embodiments, the self-cleaving polypeptide site comprises a2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen.Virol. 82:1027-1041).

TABLE 2 Exemplary 2A sites include the following sequences: SEQ IDLLNFDLLKLAGDVESNPGP NO: 26 SEQ ID TLNFDLLKLAGDVESNPGP NO: 27 SEQ IDLLKLAGDVESNPGP NO: 28 SEQ ID NFDLLKLAGDVESNPGP NO: 29 SEQ IDQLLNFDLLKLAGDVESNPGP NO: 30 SEQ ID APVKQTLNFDLLKLAGDVESNPGP NO: 31SEQ ID VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT NO: 32 SEQ IDLNFDLLKLAGDVESNPGP NO: 33 SEQ IDLLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 34 SEQ IDEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP NO: 35

In preferred embodiments, a polypeptide contemplated herein comprises aCAR polypeptide.

E. Polynucleotides

In preferred embodiments, a polynucleotide encoding one or more CARpolypeptides is provided, e.g., SEQ ID NO: 10. As used herein, the terms“polynucleotide” or “nucleic acid” refers to messenger RNA (mRNA), RNA,genomic RNA (gRNA), plus strand RNA (RNA(+)), minus strand RNA (RNA(−)),genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA.Polynucleotides include single and double stranded polynucleotides.Preferably, polynucleotides of the invention include polynucleotides orvariants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of thereference sequences described herein (see, e.g., Sequence Listing),typically where the variant maintains at least one biological activityof the reference sequence. In various illustrative embodiments, thepresent invention contemplates, in part, polynucleotides comprisingexpression vectors, viral vectors, and transfer plasmids, andcompositions, and cells comprising the same.

In particular embodiments, polynucleotides are provided by thisinvention that encode at least about 5, 10, 25, 50, 100, 150, 200, 250,300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more contiguousamino acid residues of a polypeptide of the invention, as well as allintermediate lengths. It will be readily understood that “intermediatelengths,” in this context, means any length between the quoted values,such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201,202, 203, etc.

As used herein, the terms “polynucleotide variant” and “variant” and thelike refer to polynucleotides displaying substantial sequence identitywith a reference polynucleotide sequence or polynucleotides thathybridize with a reference sequence under stringent conditions that aredefined hereinafter. These terms include polynucleotides in which one ormore nucleotides have been added or deleted, or replaced with differentnucleotides compared to a reference polynucleotide. In this regard, itis well understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains thebiological function or activity of the reference polynucleotide.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to any of the reference sequencesdescribed herein, typically where the polypeptide variant maintains atleast one biological activity of the reference polypeptide.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, “isolated polynucleotide” refers to a polynucleotidethat has been purified from the sequences which flank it in anaturally-occurring state, e.g., a DNA fragment that has been removedfrom the sequences that are normally adjacent to the fragment. An“isolated polynucleotide” also refers to a complementary DNA (cDNA), arecombinant DNA, or other polynucleotide that does not exist in natureand that has been made by the hand of man.

Terms that describe the orientation of polynucleotides include: 5′(normally the end of the polynucleotide having a free phosphate group)and 3′ (normally the end of the polynucleotide having a free hydroxyl(OH) group). Polynucleotide sequences can be annotated in the 5′ to 3′orientation or the 3′ to 5′ orientation. For DNA and mRNA, the 5′ to 3′strand is designated the “sense,” “plus,” or “coding” strand because itssequence is identical to the sequence of the premessenger (premRNA)[except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNAand mRNA, the complementary 3′ to 5′ strand which is the strandtranscribed by the RNA polymerase is designated as “template,”“antisense,” “minus,” or “non-coding” strand. As used herein, the term“reverse orientation” refers to a 5′ to 3′ sequence written in the 3′ to5′ orientation or a 3′ to 5′ sequence written in the 5′ to 3′orientation.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the complementary strand of the DNA sequence 5′ A G T C A T G3′ is 3′ T C A G T A C 5′. The latter sequence is often written as thereverse complement with the 5′ end on the left and the 3′ end on theright, 5′ C A T G A C T 3′. A sequence that is equal to its reversecomplement is said to be a palindromic sequence. Complementarity can be“partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there can be “complete” or“total” complementarity between the nucleic acids.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide, or fragment of variantthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention, for examplepolynucleotides that are optimized for human and/or primate codonselection. Further, alleles of the genes comprising the polynucleotidesequences provided herein may also be used. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides.

The term “nucleic acid cassette” as used herein refers to geneticsequences within a vector which can express a RNA, and subsequently aprotein. The nucleic acid cassette contains the gene of interest, e.g.,a CAR. The nucleic acid cassette is positionally and sequentiallyoriented within the vector such that the nucleic acid in the cassettecan be transcribed into RNA, and when necessary, translated into aprotein or a polypeptide, undergo appropriate post-translationalmodifications required for activity in the transformed cell, and betranslocated to the appropriate compartment for biological activity bytargeting to appropriate intracellular compartments or secretion intoextracellular compartments. Preferably, the cassette has its 3′ and 5′ends adapted for ready insertion into a vector, e.g., it has restrictionendonuclease sites at each end. In a preferred embodiment of theinvention, the nucleic acid cassette contains the sequence of a chimericantigen receptor used to treat a B cell malignancy. The cassette can beremoved and inserted into a plasmid or viral vector as a single unit.

In particular embodiments, polynucleotides include at least onepolynucleotide-of-interest. As used herein, the term“polynucleotide-of-interest” refers to a polynucleotide encoding apolypeptide (i.e., a polypeptide-of-interest), inserted into anexpression vector that is desired to be expressed. A vector may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides-of-interest. In certainembodiments, the polynucleotide-of-interest encodes a polypeptide thatprovides a therapeutic effect in the treatment or prevention of adisease or disorder. Polynucleotides-of-interest, and polypeptidesencoded therefrom, include both polynucleotides that encode wild-typepolypeptides, as well as functional variants and fragments thereof. Inparticular embodiments, a functional variant has at least 80%, at least90%, at least 95%, or at least 99% identity to a corresponding wild-typereference polynucleotide or polypeptide sequence. In certainembodiments, a functional variant or fragment has at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of a biologicalactivity of a corresponding wild-type polypeptide.

In one embodiment, the polynucleotide-of-interest does not encode apolypeptide but serves as a template to transcribe miRNA, siRNA, orshRNA, ribozyme, or other inhibitory RNA. In various other embodiments,a polynucleotide comprises a polynucleotide-of-interest encoding a CARand one or more additional polynucleotides-of-interest including but notlimited to an inhibitory nucleic acid sequence including, but notlimited to: an siRNA, an miRNA, an shRNA, and a ribozyme.

As used herein, the terms “siRNA” or “short interfering RNA” refer to ashort polynucleotide sequence that mediates a process ofsequence-specific post-transcriptional gene silencing, translationalinhibition, transcriptional inhibition, or epigenetic RNAi in animals(Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391,806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999,Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; andStrauss, 1999, Science, 286, 886). In certain embodiments, an siRNAcomprises a first strand and a second strand that have the same numberof nucleosides; however, the first and second strands are offset suchthat the two terminal nucleosides on the first and second strands arenot paired with a residue on the complimentary strand. In certaininstances, the two nucleosides that are not paired are thymidineresides. The siRNA should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the siRNA, or a fragment thereof, can mediate down regulation ofthe target gene. Thus, an siRNA includes a region which is at leastpartially complementary to the target RNA. It is not necessary thatthere be perfect complementarity between the siRNA and the target, butthe correspondence must be sufficient to enable the siRNA, or a cleavageproduct thereof, to direct sequence specific silencing, such as by RNAicleavage of the target RNA. Complementarity, or degree of homology withthe target strand, is most critical in the antisense strand. Whileperfect complementarity, particularly in the antisense strand, is oftendesired, some embodiments include one or more, but preferably 10, 8, 6,5, 4, 3, 2, or fewer mismatches with respect to the target RNA. Themismatches are most tolerated in the terminal regions, and if presentare preferably in a terminal region or regions, e.g., within 6, 5, 4, or3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need onlybe sufficiently complementary with the antisense strand to maintain theoverall double-strand character of the molecule.

In addition, an siRNA may be modified or include nucleoside analogs.Single stranded regions of an siRNA may be modified or includenucleoside analogs, e.g., the unpaired region or regions of a hairpinstructure, e.g., a region which links two complementary regions, canhave modifications or nucleoside analogs. Modification to stabilize oneor more 3′- or 5′-terminus of an siRNA, e.g., against exonucleases, orto favor the antisense siRNA agent to enter into RISC are also useful.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis. Each strand of an siRNA can be equal to or less than 30, 25,24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred siRNAs have a duplex regionof 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one ormore overhangs of 2-3 nucleotides, preferably one or two 3′ overhangs,of 2-3 nucleotides.

As used herein, the terms “miRNA” or “microRNA” refer to smallnon-coding RNAs of 20-22 nucleotides, typically excised from ˜70nucleotide fold-back RNA precursor structures known as pre-miRNAs.miRNAs negatively regulate their targets in one of two ways depending onthe degree of complementarity between the miRNA and the target. First,miRNAs that bind with perfect or nearly perfect complementarity toprotein-coding mRNA sequences induce the RNA-mediated interference(RNAi) pathway. miRNAs that exert their regulatory effects by binding toimperfect complementary sites within the 3′ untranslated regions (UTRs)of their mRNA targets, repress target-gene expressionpost-transcriptionally, apparently at the level of translation, througha RISC complex that is similar to, or possibly identical with, the onethat is used for the RNAi pathway. Consistent with translationalcontrol, miRNAs that use this mechanism reduce the protein levels oftheir target genes, but the mRNA levels of these genes are onlyminimally affected. miRNAs encompass both naturally occurring miRNAs aswell as artificially designed miRNAs that can specifically target anymRNA sequence. For example, in one embodiment, the skilled artisan candesign short hairpin RNA constructs expressed as human miRNA (e.g.,miR-30 or miR-21) primary transcripts. This design adds a Droshaprocessing site to the hairpin construct and has been shown to greatlyincrease knockdown efficiency (Pusch et al., 2004). The hairpin stemconsists of 22-nt of dsRNA (e.g., antisense has perfect complementarityto desired target) and a 15-19-nt loop from a human miR. Adding the miRloop and miR30 flanking sequences on either or both sides of the hairpinresults in greater than 10-fold increase in Drosha and Dicer processingof the expressed hairpins when compared with conventional shRNA designswithout microRNA. Increased Drosha and Dicer processing translates intogreater siRNA/miRNA production and greater potency for expressedhairpins.

As used herein, the terms “shRNA” or “short hairpin RNA” refer todouble-stranded structure that is formed by a single self-complementaryRNA strand. shRNA constructs containing a nucleotide sequence identicalto a portion, of either coding or non-coding sequence, of the targetgene are preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Greater than 90%sequence identity, or even 100% sequence identity, between theinhibitory RNA and the portion of the target gene is preferred. Incertain preferred embodiments, the length of the duplex-forming portionof an shRNA is at least 20, 21 or 22 nucleotides in length, e.g.,corresponding in size to RNA products produced by Dicer-dependentcleavage. In certain embodiments, the shRNA construct is at least 25,50, 100, 200, 300 or 400 bases in length. In certain embodiments, theshRNA construct is 400-800 bases in length. shRNA constructs are highlytolerant of variation in loop sequence and loop size.\

As used herein, the term “ribozyme” refers to a catalytically active RNAmolecule capable of site-specific cleavage of target mRNA. Severalsubtypes have been described, e.g., hammerhead and hairpin ribozymes.Ribozyme catalytic activity and stability can be improved bysubstituting deoxyribonucleotides for ribonucleotides at noncatalyticbases. While ribozymes that cleave mRNA at site-specific recognitionsequences can be used to destroy particular mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA has thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art.

A preferred method of delivery of a polynucleotide-of-interest thatcomprises an siRNA, an miRNA, an shRNA, or a ribozyme comprises one ormore regulatory sequences, such as, for example, a strong constitutivepol III, e.g., human U6 snRNA promoter, the mouse U6 snRNA promoter, thehuman and mouse H1 RNA promoter and the human tRNA-val promoter, or astrong constitutive pol II promoter, as described elsewhere herein.

The polynucleotides of the present invention, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art, such that theiroverall length may vary considerably. It is therefore contemplated thata polynucleotide fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated and/or expressed using anyof a variety of well-established techniques known and available in theart. In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, can be inserted into appropriate vector.Examples of vectors are plasmid, autonomously replicating sequences, andtransposable elements. Additional exemplary vectors include, withoutlimitation, plasmids, phagemids, cosmids, artificial chromosomes such asyeast artificial chromosome (YAC), bacterial artificial chromosome(BAC), or P1-derived artificial chromosome (PAC), bacteriophages such aslambda phage or M13 phage, and animal viruses. Examples of categories ofanimal viruses useful as vectors include, without limitation, retrovirus(including lentivirus), adenovirus, adeno-associated virus, herpesvirus(e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, andpapovavirus (e.g., SV40). Examples of expression vectors are pClneovectors (Promega) for expression in mammalian cells; pLenti4/V5-DEST™,pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells. Inparticular embodiments, the coding sequences of the chimeric proteinsdisclosed herein can be ligated into such expression vectors for theexpression of the chimeric protein in mammalian cells.

In one embodiment, a vector encoding a CAR contemplated herein comprisesthe polynucleotide sequence set forth in SEQ ID NO: 36.

In particular embodiments, the vector is an episomal vector or a vectorthat is maintained extrachromosomally. As used herein, the term“episomal” refers to a vector that is able to replicate withoutintegration into host's chromosomal DNA and without gradual loss from adividing host cell also meaning that said vector replicatesextrachromosomally or episomally. The vector is engineered to harbor thesequence coding for the origin of DNA replication or “ori” from alymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40,a bovine papilloma virus, or a yeast, specifically a replication originof a lymphotrophic herpes virus or a gamma herpesvirus corresponding tooriP of EBV. In a particular aspect, the lymphotrophic herpes virus maybe Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV),Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein Barrvirus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examplesof a gamma herpesvirus. Typically, the host cell comprises the viralreplication transactivator protein that activates the replication.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of the vector—originof replication, selection cassettes, promoters, enhancers, translationinitiation signals (Shine Dalgarno sequence or Kozak sequence) introns,a polyadenylation sequence, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including ubiquitous promotersand inducible promoters may be used.

In particular embodiments, a vector for use in practicing the inventionincluding, but not limited to expression vectors and viral vectors, willinclude exogenous, endogenous, or heterologous control sequences such aspromoters and/or enhancers. An “endogenous” control sequence is onewhich is naturally linked with a given gene in the genome. An“exogenous” control sequence is one which is placed in juxtaposition toa gene by means of genetic manipulation (i.e., molecular biologicaltechniques) such that transcription of that gene is directed by thelinked enhancer/promoter. A “heterologous” control sequence is anexogenous sequence that is from a different species than the cell beinggenetically manipulated.

The term “promoter” as used herein refers to a recognition site of apolynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNApolymerase initiates and transcribes polynucleotides operably linked tothe promoter. In particular embodiments, promoters operative inmammalian cells comprise an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated and/oranother sequence found 70 to 80 bases upstream from the start oftranscription, a CNCAAT region where N may be any nucleotide.

The term “enhancer” refers to a segment of DNA which contains sequencescapable of providing enhanced transcription and in some instances canfunction independent of their orientation relative to another controlsequence. An enhancer can function cooperatively or additively withpromoters and/or other enhancer elements. The term “promoter/enhancer”refers to a segment of DNA which contains sequences capable of providingboth promoter and enhancer functions.

The term “operably linked”, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. In one embodiment, the term refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, and/or enhancer) and a second polynucleotidesequence, e.g., a polynucleotide-of-interest, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

As used herein, the term “constitutive expression control sequence”refers to a promoter, enhancer, or promoter/enhancer that continually orcontinuously allows for transcription of an operably linked sequence. Aconstitutive expression control sequence may be a “ubiquitous” promoter,enhancer, or promoter/enhancer that allows expression in a wide varietyof cell and tissue types or a “cell specific,” “cell type specific,”“cell lineage specific,” or “tissue specific” promoter, enhancer, orpromoter/enhancer that allows expression in a restricted variety of celland tissue types, respectively.

Illustrative ubiquitous expression control sequences suitable for use inparticular embodiments of the invention include, but are not limited to,a cytomegalovirus (CMV) immediate early promoter, a viral simian virus40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV)LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters fromvaccinia virus, an elongation factor 1-alpha (EF1a) promoter, earlygrowth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL),Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translationinitiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions etal., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, a β-actin promoter and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer-binding site substituted (MND) promoter(Challita et al., J Virol. 69(2):748-55 (1995)).

In one embodiment, a vector of the invention comprises a MND promoter.

In one embodiment, a vector of the invention comprises an EF1a promotercomprising the first intron of the human EF1a gene.

In one embodiment, a vector of the invention comprises an EF1a promoterthat lacks the first intron of the human EF1a gene.

In a particular embodiment, it may be desirable to express apolynucleotide comprising a CAR from a T cell specific promoter.

As used herein, “conditional expression” may refer to any type ofconditional expression including, but not limited to, inducibleexpression; repressible expression; expression in cells or tissueshaving a particular physiological, biological, or disease state, etc.This definition is not intended to exclude cell type or tissue specificexpression. Certain embodiments of the invention provide conditionalexpression of a polynucleotide-of-interest, e.g., expression iscontrolled by subjecting a cell, tissue, organism, etc., to a treatmentor condition that causes the polynucleotide to be expressed or thatcauses an increase or decrease in expression of the polynucleotideencoded by the polynucleotide-of-interest.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved by using a site specific DNArecombinase. According to certain embodiments of the invention thevector comprises at least one (typically two) site(s) for recombinationmediated by a site specific recombinase. As used herein, the terms“recombinase” or “site specific recombinase” include excisive orintegrative proteins, enzymes, co-factors or associated proteins thatare involved in recombination reactions involving one or morerecombination sites (e.g., two, three, four, five, seven, ten, twelve,fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins(see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), ormutants, derivatives (e.g., fusion proteins containing the recombinationprotein sequences or fragments thereof), fragments, and variantsthereof. Illustrative examples of recombinases suitable for use inparticular embodiments of the present invention include, but are notlimited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

The vectors may comprise one or more recombination sites for any of awide variety of site specific recombinases. It is to be understood thatthe target site for a site specific recombinase is in addition to anysite(s) required for integration of a vector, e.g., a retroviral vectoror lentiviral vector. As used herein, the terms “recombinationsequence,” “recombination site,” or “site specific recombination site”refer to a particular nucleic acid sequence to which a recombinaserecognizes and binds.

For example, one recombination site for Cre recombinase is loxP which isa 34 base pair sequence comprising two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology5:521-527 (1994)). Other exemplary loxP sites include, but are notlimited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171(Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al.,2002), lox7l (Albert et al., 1995), and lox66 (Albert et al., 1995).

Suitable recognition sites for the FLP recombinase include, but are notlimited to: FRT (McLeod, et al., 1996), F₁, F₂, F₃ (Schlake and Bode,1994), F₄, F₅ (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988),FRT(RE) (Senecoff et al., 1988).

Other examples of recognition sequences are the attB, attP, attL, andattR sequences, which are recognized by the recombinase enzyme λIntegrase, e.g., phi-c31. The φC31 SSR mediates recombination onlybetween the heterotypic sites attB (34 bp in length) and attP (39 bp inlength) (Groth et al., 2000). attB and attP, named for the attachmentsites for the phage integrase on the bacterial and phage genomes,respectively, both contain imperfect inverted repeats that are likelybound by φC31 homodimers (Groth et al., 2000). The product sites, attLand attR, are effectively inert to further φC31-mediated recombination(Belteki et al., 2003), making the reaction irreversible. For catalyzinginsertions, it has been found that attB-bearing DNA inserts into agenomic attP site more readily than an attP site into a genomic attBsite (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, typicalstrategies position by homologous recombination an attP-bearing “dockingsite” into a defined locus, which is then partnered with an attB-bearingincoming sequence for insertion.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson andKaminski. 1995. RNA 1(10):985-1000. In particular embodiments, thevectors contemplated by the invention, include one or morepolynucleotides-of-interest that encode one or more polypeptides. Inparticular embodiments, to achieve efficient translation of each of theplurality of polypeptides, the polynucleotide sequences can be separatedby one or more IRES sequences or polynucleotide sequences encodingself-cleaving polypeptides.

As used herein, the term “Kozak sequence” refers to a short nucleotidesequence that greatly facilitates the initial binding of mRNA to thesmall subunit of the ribosome and increases translation. The consensusKozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak,1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res.15(20):8125-48). In particular embodiments, the vectors contemplated bythe invention, comprise polynucleotides that have a consensus Kozaksequence and that encode a desired polypeptide, e.g., a CAR.

In some embodiments of the invention, a polynucleotide or cell harboringthe polynucleotide utilizes a suicide gene, including an induciblesuicide gene to reduce the risk of direct toxicity and/or uncontrolledproliferation. In specific aspects, the suicide gene is not immunogenicto the host harboring the polynucleotide or cell. A certain example of asuicide gene that may be used is caspase-9 or caspase-8 or cytosinedeaminase. Caspase-9 can be activated using a specific chemical inducerof dimerization (CID).

In certain embodiments, vectors comprise gene segments that cause theimmune effector cells of the invention, e.g., T cells, to be susceptibleto negative selection in vivo. By “negative selection” is meant that theinfused cell can be eliminated as a result of a change in the in vivocondition of the individual. The negative selectable phenotype mayresult from the insertion of a gene that confers sensitivity to anadministered agent, for example, a compound. Negative selectable genesare known in the art, and include, inter alia the following: the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell 11:223, 1977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, genetically modified immune effector cells, such asT cells, comprise a polynucleotide further comprising a positive markerthat enables the selection of cells of the negative selectable phenotypein vitro. The positive selectable marker may be a gene which, upon beingintroduced into the host cell expresses a dominant phenotype permittingpositive selection of cells carrying the gene. Genes of this type areknown in the art, and include, inter alia, hygromycin-Bphosphotransferase gene (hph) which confers resistance to hygromycin B,the amino glycoside phosphotransferase gene (neo or aph) from Tn5 whichcodes for resistance to the antibiotic G418, the dihydrofolate reductase(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drugresistance (MDR) gene.

Preferably, the positive selectable marker and the negative selectableelement are linked such that loss of the negative selectable elementnecessarily also is accompanied by loss of the positive selectablemarker. Even more preferably, the positive and negative selectablemarkers are fused so that loss of one obligatorily leads to loss of theother. An example of a fused polynucleotide that yields as an expressionproduct a polypeptide that confers both the desired positive andnegative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See Lupton S. D., et al, Mol. and Cell. Biology 11:3374-3378, 1991. In addition, in preferred embodiments, thepolynucleotides of the invention encoding the chimeric receptors are inretroviral vectors containing the fused gene, particularly those thatconfer hygromycin B resistance for positive selection in vitro, andganciclovir sensitivity for negative selection in vivo, for example theHyTK retroviral vector described in Lupton, S. D. et al. (1991), supra.See also the publications of PCT US91/08442 and PCT/US94/05601, by S. D.Lupton, describing the use of bifunctional selectable fusion genesderived from fusing a dominant positive selectable markers with negativeselectable markers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, nco, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Especially preferred markers are bifunctional selectable fusion geneswherein the positive selectable marker is derived from hph or neo, andthe negative selectable marker is derived from cytosine deaminase or aTK gene or selectable marker. Inducible Suicide Genes

F. Viral Vectors

In particular embodiments, a cell (e.g., an immune effector cell) istransduced with a retroviral vector, e.g., a lentiviral vector, encodinga CAR. For example, an immune effector cell is transduced with a vectorencoding a CAR that comprises a murine anti-BCMA antibody or antigenbinding fragment thereof that binds a BCMA polypeptide, with anintracellular signaling domain of CD3ζ, CD28, 4-1BB, Ox40, or anycombinations thereof. Thus, these transduced cells can elicit aCAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery (Miller, 2000, Nature.357: 455-460). In particular embodiments, a retrovirus is used todeliver a polynucleotide encoding a chimeric antigen receptor (CAR) to acell. As used herein, the term “retrovirus” refers to an RNA virus thatreverse transcribes its genomic RNA into a linear double-stranded DNAcopy and subsequently covalently integrates its genomic DNA into a hostgenome. Once the virus is integrated into the host genome, it isreferred to as a “provirus.” The provirus serves as a template for RNApolymerase II and directs the expression of RNA molecules which encodethe structural proteins and enzymes needed to produce new viralparticles.

Illustrative retroviruses suitable for use in particular embodiments,include, but are not limited to: Moloney murine leukemia virus (M-MuLV),Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) andlentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include, but are notlimited to: HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred. In particular embodiments, a lentivirus is used to deliver apolynucleotide comprising a CAR to a cell.

Retroviral vectors and more particularly lentiviral vectors may be usedin practicing particular embodiments of the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively.

The term “vector” is used herein to refer to a nucleic acid moleculecapable transferring or transporting another nucleic acid molecule. Thetransferred nucleic acid is generally linked to, e.g., inserted into,the vector nucleic acid molecule. A vector may include sequences thatdirect autonomous replication in a cell, or may include sequencessufficient to allow integration into host cell DNA. Useful vectorsinclude, for example, plasmids (e.g., DNA plasmids or RNA plasmids),transposons, cosmids, bacterial artificial chromosomes, and viralvectors. Useful viral vectors include, e.g., replication defectiveretroviruses and lentiviruses.

As will be evident to one of skill in the art, the term “viral vector”is widely used to refer either to a nucleic acid molecule (e.g., atransfer plasmid) that includes virus-derived nucleic acid elements thattypically facilitate transfer of the nucleic acid molecule orintegration into the genome of a cell or to a viral particle thatmediates nucleic acid transfer. Viral particles will typically includevarious viral components and sometimes also host cell components inaddition to nucleic acid(s).

The term viral vector may refer either to a virus or viral particlecapable of transferring a nucleic acid into a cell or to the transferrednucleic acid itself. Viral vectors and transfer plasmids containstructural and/or functional genetic elements that are primarily derivedfrom a virus. The term “retroviral vector” refers to a viral vector orplasmid containing structural and functional genetic elements, orportions thereof, that are primarily derived from a retrovirus. The term“lentiviral vector” refers to a viral vector or plasmid containingstructural and functional genetic elements, or portions thereof,including LTRs that are primarily derived from a lentivirus. The term“hybrid vector” refers to a vector, LTR or other nucleic acid containingboth retroviral, e.g., lentiviral, sequences and non-lentiviral viralsequences. In one embodiment, a hybrid vector refers to a vector ortransfer plasmid comprising retroviral e.g., lentiviral, sequences forreverse transcription, replication, integration and/or packaging.

In particular embodiments, the terms “lentiviral vector,” “lentiviralexpression vector” may be used to refer to lentiviral transfer plasmidsand/or infectious lentiviral particles. Where reference is made hereinto elements such as cloning sites, promoters, regulatory elements,heterologous nucleic acids, etc., it is to be understood that thesequences of these elements are present in RNA form in the lentiviralparticles of the invention and are present in DNA form in the DNAplasmids of the invention.

At each end of the provirus are structures called “long terminalrepeats” or “LTRs.” The term “long terminal repeat (LTR)” refers todomains of base pairs located at the ends of retroviral DNAs which, intheir natural sequence context, are direct repeats and contain U3, R andU5 regions. LTRs generally provide functions fundamental to theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. The LTRcontains numerous regulatory signals including transcriptional controlelements, polyadenylation signals and sequences needed for replicationand integration of the viral genome. The viral LTR is divided into threeregions called U3, R and U5. The U3 region contains the enhancer andpromoter elements. The U5 region is the sequence between the primerbinding site and the R region and contains the polyadenylation sequence.The R (repeat) region is flanked by the U3 and U5 regions. The LTRcomposed of U3, R and U5 regions and appears at both the 5′ and 3′ endsof the viral genome. Adjacent to the 5′ LTR are sequences necessary forreverse transcription of the genome (the tRNA primer binding site) andfor efficient packaging of viral RNA into particles (the Psi site).

As used herein, the term “packaging signal” or “packaging sequence”refers to sequences located within the retroviral genome which arerequired for insertion of the viral RNA into the viral capsid orparticle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4;pp. 2101-2109. Several retroviral vectors use the minimal packagingsignal (also referred to as the psi [Ψ] sequence) needed forencapsidation of the viral genome. Thus, as used herein, the terms“packaging sequence,” “packaging signal,” “psi” and the symbol “Ψ,” areused in reference to the non-coding sequence required for encapsidationof retroviral RNA strands during viral particle formation.

In various embodiments, vectors comprise modified 5′ LTR and/or 3′ LTRs.Either or both of the LTR may comprise one or more modificationsincluding, but not limited to, one or more deletions, insertions, orsubstitutions. Modifications of the 3′ LTR are often made to improve thesafety of lentiviral or retroviral systems by rendering virusesreplication-defective. As used herein, the term “replication-defective”refers to virus that is not capable of complete, effective replicationsuch that infective virions are not produced (e.g.,replication-defective lentiviral progeny). The term“replication-competent” refers to wild-type virus or mutant virus thatis capable of replication, such that viral replication of the virus iscapable of producing infective virions (e.g., replication-competentlentiviral progeny).

“Self-inactivating” (SIN) vectors refers to replication-defectivevectors, e.g., retroviral or lentiviral vectors, in which the right (3′)LTR enhancer-promoter region, known as the U3 region, has been modified(e.g., by deletion or substitution) to prevent viral transcriptionbeyond the first round of viral replication. This is because the right(3′) LTR U3 region is used as a template for the left (5′) LTR U3 regionduring viral replication and, thus, the viral transcript cannot be madewithout the U3 enhancer-promoter. In a further embodiment of theinvention, the 3′ LTR is modified such that the U5 region is replaced,for example, with an ideal poly(A) sequence. It should be noted thatmodifications to the LTRs such as modifications to the 3′ LTR, the 5′LTR, or both 3′ and 5′ LTRs, are also included in the invention.

An additional safety enhancement is provided by replacing the U3 regionof the 5′ LTR with a heterologous promoter to drive transcription of theviral genome during production of viral particles. Examples ofheterologous promoters which can be used include, for example, viralsimian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV)(e.g., immediate early), Moloney murine leukemia virus (MoMLV), Roussarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase)promoters. Typical promoters are able to drive high levels oftranscription in a Tat-independent manner. This replacement reduces thepossibility of recombination to generate replication-competent virusbecause there is no complete U3 sequence in the virus production system.In certain embodiments, the heterologous promoter has additionaladvantages in controlling the manner in which the viral genome istranscribed. For example, the heterologous promoter can be inducible,such that transcription of all or part of the viral genome will occuronly when the induction factors are present. Induction factors include,but are not limited to, one or more chemical compounds or thephysiological conditions such as temperature or pH, in which the hostcells are cultured.

In some embodiments, viral vectors comprise a TAR element. The term“TAR” refers to the “trans-activation response” genetic element locatedin the R region of lentiviral (e.g., HIV) LTRs. This element interactswith the lentiviral trans-activator (tat) genetic element to enhanceviral replication. However, this element is not required in embodimentswherein the U3 region of the 5′ LTR is replaced by a heterologouspromoter.

The “R region” refers to the region within retroviral LTRs beginning atthe start of the capping group (i.e., the start of transcription) andending immediately prior to the start of the poly A tract. The R regionis also defined as being flanked by the U3 and U5 regions. The R regionplays a role during reverse transcription in permitting the transfer ofnascent DNA from one end of the genome to the other.

As used herein, the term “FLAP element” refers to a nucleic acid whosesequence includes the central polypurine tract and central terminationsequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. SuitableFLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, etal., 2000, Cell, 101:173. During HIV-1 reverse transcription, centralinitiation of the plus-strand DNA at the central polypurine tract (cPPT)and central termination at the central termination sequence (CTS) leadto the formation of a three-stranded DNA structure: the HIV-1 centralDNA flap. While not wishing to be bound by any theory, the DNA flap mayact as a cis-active determinant of lentiviral genome nuclear importand/or may increase the titer of the virus. In particular embodiments,the retroviral or lentiviral vector backbones comprise one or more FLAPelements upstream or downstream of the heterologous genes of interest inthe vectors. For example, in particular embodiments a transfer plasmidincludes a FLAP element. In one embodiment, a vector of the inventioncomprises a FLAP element isolated from HIV-1.

In one embodiment, retroviral or lentiviral transfer vectors compriseone or more export elements. The term “export element” refers to acis-acting post-transcriptional regulatory element which regulates thetransport of an RNA transcript from the nucleus to the cytoplasm of acell. Examples of RNA export elements include, but are not limited to,the human immunodeficiency virus (HIV) rev response element (RRE) (seee.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991.Cell 58: 423), and the hepatitis B virus post-transcriptional regulatoryelement (HPRE). Generally, the RNA export element is placed within the3′ UTR of a gene, and can be inserted as one or multiple copies.

In particular embodiments, expression of heterologous sequences in viralvectors is increased by incorporating posttranscriptional regulatoryelements, efficient polyadenylation sites, and optionally, transcriptiontermination signals into the vectors. A variety of posttranscriptionalregulatory elements can increase expression of a heterologous nucleicacid at the protein, e.g., woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886);the posttranscriptional regulatory element present in hepatitis B virus(HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu etal., 1995, Genes Dev., 9:1766). In particular embodiments, vectors ofthe invention comprise a posttranscriptional regulatory element such asa WPRE or HPRE

In particular embodiments, vectors of the invention lack or do notcomprise a posttranscriptional regulatory element (PTE) such as a WPREor HPRE because in some instances these elements increase the risk ofcellular transformation and/or do not substantially or significantlyincrease the amount of mRNA transcript or increase mRNA stability.Therefore, in some embodiments, vectors of the invention lack or do notcomprise a PTE. In other embodiments, vectors of the invention lack ordo not comprise a WPRE or HPRE as an added safety measure.

Elements directing the efficient termination and polyadenylation of theheterologous nucleic acid transcripts increases heterologous geneexpression. Transcription termination signals are generally founddownstream of the polyadenylation signal. In particular embodiments,vectors comprise a polyadenylation sequence 3′ of a polynucleotideencoding a polypeptide to be expressed. The term “polyA site” or “polyAsequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript by RNApolymerase II. Polyadenylation sequences can promote mRNA stability byaddition of a polyA tail to the 3′ end of the coding sequence and thus,contribute to increased translational efficiency. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.Illustrative examples of polyA signals that can be used in a vector ofthe invention, includes an ideal polyA sequence (e.g., AATAAA, ATTAAA,AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a rabbitβ-globin polyA sequence (rβgpA), or another suitable heterologous orendogenous polyA sequence known in the art.

In certain embodiments, a retroviral or lentiviral vector furthercomprises one or more insulator elements. Insulators elements maycontribute to protecting lentivirus-expressed sequences, e.g.,therapeutic polypeptides, from integration site effects, which may bemediated by cis-acting elements present in genomic DNA and lead toderegulated expression of transferred sequences (i.e., position effect;see, e.g., Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA,99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In someembodiments, transfer vectors comprise one or more insulator element the3′ LTR and upon integration of the provirus into the host genome, theprovirus comprises the one or more insulators at both the 5′ LTR or 3′LTR, by virtue of duplicating the 3′ LTR. Suitable insulators for use inthe invention include, but are not limited to, the chicken β-globininsulator (see Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS94:575; and Bell et al., 1999. Cell 98:387, incorporated by referenceherein). Examples of insulator elements include, but are not limited to,an insulator from an β-globin locus, such as chicken HS4.

According to certain specific embodiments of the invention, most or allof the viral vector backbone sequences are derived from a lentivirus,e.g., HIV-1. However, it is to be understood that many different sourcesof retroviral and/or lentiviral sequences can be used, or combined andnumerous substitutions and alterations in certain of the lentiviralsequences may be accommodated without impairing the ability of atransfer vector to perform the functions described herein. Moreover, avariety of lentiviral vectors are known in the art, see Naldini et al.,(1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998,U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which may be adapted toproduce a viral vector or transfer plasmid of the present invention.

In various embodiments, the vectors of the invention comprise a promoteroperably linked to a polynucleotide encoding a CAR polypeptide. Thevectors may have one or more LTRs, wherein either LTR comprises one ormore modifications, such as one or more nucleotide substitutions,additions, or deletions. The vectors may further comprise one of moreaccessory elements to increase transduction efficiency (e.g., acPPT/FLAP), viral packaging (e.g., a Psi (Ψ) packaging signal, RRE),and/or other elements that increase therapeutic gene expression (e.g.,poly (A) sequences), and may optionally comprise a WPRE or HPRE.

In a particular embodiment, the transfer vector of the inventioncomprises a left (5′) retroviral LTR; a central polypurine tract/DNAflap (cPPT/FLAP); a retroviral export element; a promoter active in a Tcell, operably linked to a polynucleotide encoding CAR polypeptidecontemplated herein; and a right (3′) retroviral LTR; and optionally aWPRE or HPRE.

In a particular embodiment, the transfer vector of the inventioncomprises a left (5′) retroviral LTR; a retroviral export element; apromoter active in a T cell, operably linked to a polynucleotideencoding CAR polypeptide contemplated herein; a right (3′) retroviralLTR; and a poly (A) sequence; and optionally a WPRE or HPRE. In anotherparticular embodiment, the invention provides a lentiviral vectorcomprising: a left (5′) LTR; a cPPT/FLAP; an RRE; a promoter active in aT cell, operably linked to a polynucleotide encoding CAR polypeptidecontemplated herein; a right (3′) LTR; and a polyadenylation sequence;and optionally a WPRE or HPRE.

In a certain embodiment, the invention provides a lentiviral vectorcomprising: a left (5′) HIV-1 LTR; a Psi (Ψ) packaging signal; acPPT/FLAP; an RRE; a promoter active in a T cell, operably linked to apolynucleotide encoding CAR polypeptide contemplated herein; a right(3′) self-inactivating (SIN) HIV-1 LTR; and a rabbit β-globinpolyadenylation sequence; and optionally a WPRE or HPRE.

In another embodiment, the invention provides a vector comprising: atleast one LTR; a central polypurine tract/DNA flap (cPPT/FLAP); aretroviral export element; and a promoter active in a T cell, operablylinked to a polynucleotide encoding CAR polypeptide contemplated herein;and optionally a WPRE or HPRE.

In particular embodiment, the present invention provides a vectorcomprising at least one LTR; a cPPT/FLAP; an RRE; a promoter active in aT cell, operably linked to a polynucleotide encoding CAR polypeptidecontemplated herein; and a polyadenylation sequence; and optionally aWPRE or HPRE.

In a certain embodiment, the present invention provides at least one SINHIV-1 LTR; a Psi (Ψ) packaging signal; a cPPT/FLAP; an RRE; a promoteractive in a T cell, operably linked to a polynucleotide encoding CARpolypeptide contemplated herein; and a rabbit β-globin polyadenylationsequence; and optionally a WPRE or HPRE.

In various embodiments, the vector is an integrating viral vector.

In various other embodiments, the vector is an episomal ornon-integrating viral vector.

In various embodiments, vectors contemplated herein, comprisenon-integrating or integration defective retrovirus. In one embodiment,an “integration defective” retrovirus or lentivirus refers to retrovirusor lentivirus having an integrase that lacks the capacity to integratethe viral genome into the genome of the host cells. In variousembodiments, the integrase protein is mutated to specifically decreaseits integrase activity. Integration-incompetent lentiviral vectors areobtained by modifying the pol gene encoding the integrase protein,resulting in a mutated pol gene encoding an integrative deficientintegrase. Such integration-incompetent viral vectors have beendescribed in patent application WO 2006/010834, which is hereinincorporated by reference in its entirety.

Illustrative mutations in the HIV-1 pol gene suitable to reduceintegrase activity include, but are not limited to: H12N, H12C, H16C,H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A,E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E,K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A,K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A,Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A,R262A, R263A and K264H.

Illustrative mutations in the HIV-1 pol gene suitable to reduceintegrase activity include, but are not limited to: D64E, D64V, E92K,D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E,K156A, E157A, K159E, K159A, W235F, and W235E.

In a particular embodiment, an integrase comprises a mutation in one ormore of amino acids, D64, D116 or E152. In one embodiment, an integrasecomprises a mutation in the amino acids, D64, D116 and E152. In aparticular embodiment, a defective HIV-1 integrase comprises a D64Vmutation.

A “host cell” includes cells electroporated, transfected, infected, ortransduced in vivo, ex vivo, or in vitro with a recombinant vector or apolynucleotide of the invention. Host cells may include packaging cells,producer cells, and cells infected with viral vectors. In particularembodiments, host cells infected with viral vector of the invention areadministered to a subject in need of therapy. In certain embodiments,the term “target cell” is used interchangeably with host cell and refersto transfected, infected, or transduced cells of a desired cell type. Inpreferred embodiments, the target cell is a T cell.

Large scale viral particle production is often necessary to achieve areasonable viral titer. Viral particles are produced by transfecting atransfer vector into a packaging cell line that comprises viralstructural and/or accessory genes, e.g., gag, pol, env, tat, rev, vif,vpr, vpu, vpx, or nef genes or other retroviral genes.

As used herein, the term “packaging vector” refers to an expressionvector or viral vector that lacks a packaging signal and comprises apolynucleotide encoding one, two, three, four or more viral structuraland/or accessory genes. Typically, the packaging vectors are included ina packaging cell, and are introduced into the cell via transfection,transduction or infection. Methods for transfection, transduction orinfection are well known by those of skill in the art. Aretroviral/lentiviral transfer vector of the present invention can beintroduced into a packaging cell line, via transfection, transduction orinfection, to generate a producer cell or cell line. The packagingvectors of the present invention can be introduced into human cells orcell lines by standard methods including, e.g., calcium phosphatetransfection, lipofection or electroporation. In some embodiments, thepackaging vectors are introduced into the cells together with a dominantselectable marker, such as neomycin, hygromycin, puromycin, blastocidin,zeocin, thymidine kinase, DHFR, Gln synthetase or ADA, followed byselection in the presence of the appropriate drug and isolation ofclones. A selectable marker gene can be linked physically to genesencoding by the packaging vector, e.g., by IRES or self-cleaving viralpeptides.

Viral envelope proteins (env) determine the range of host cells whichcan ultimately be infected and transformed by recombinant retrovirusesgenerated from the cell lines. In the case of lentiviruses, such asHIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120.Preferably, the viral env proteins expressed by packaging cells of theinvention are encoded on a separate vector from the viral gag and polgenes, as has been previously described.

Illustrative examples of retroviral-derived env genes which can beemployed in the invention include, but are not limited to: MLVenvelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV(Fowl plague virus), and influenza virus envelopes. Similarly, genesencoding envelopes from RNA viruses (e.g., RNA virus families ofPicornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae,Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae,Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae,Retroviridae) as well as from the DNA viruses (families ofHepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae,Herpesviridae, Poxyiridae, and Iridoviridae) may be utilized.Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies,ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2,AEV, AMV, CT10, and EIAV.

In other embodiments, envelope proteins for pseudotyping a virus ofpresent invention include, but are not limited to any of the followingvirus: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu),Influenza B, Influenza C virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, anyvirus of the Norwalk virus group, enteric adenoviruses, parvovirus,Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such asrabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Europeanbat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus,Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplexvirus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Barvirus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8,Human immunodeficiency virus (HIV), papilloma virus, murinegammaherpesvirus, Arenaviruses such as Argentine hemorrhagic fevervirus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagicfever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus,Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviridiaesuch as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagicfever with renal syndrome causing virus, Rift Valley fever virus,Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburghemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus,Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus andParamyxoviridae such as Hendra virus and Nipah virus, variola major andvariola minor (smallpox), alphaviruses such as Venezuelan equineencephalitis virus, eastern equine encephalitis virus, western equineencephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nilevirus, any encephaliltis causing virus.

In one embodiment, the invention provides packaging cells which producerecombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-Gglycoprotein.

The terms “pseudotype” or “pseudotyping” as used herein, refer to avirus whose viral envelope proteins have been substituted with those ofanother virus possessing preferable characteristics. For example, HIVcan be pseudotyped with vesicular stomatitis virus G-protein (VSV-G)envelope proteins, which allows HIV to infect a wider range of cellsbecause HIV envelope proteins (encoded by the env gene) normally targetthe virus to CD4+ presenting cells. In a preferred embodiment of theinvention, lentiviral envelope proteins are pseudotyped with VSV-G. Inone embodiment, the invention provides packaging cells which producerecombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-Genvelope glycoprotein.

As used herein, the term “packaging cell lines” is used in reference tocell lines that do not contain a packaging signal, but do stably ortransiently express viral structural proteins and replication enzymes(e.g., gag, pol and env) which are necessary for the correct packagingof viral particles. Any suitable cell line can be employed to preparepackaging cells of the invention. Generally, the cells are mammaliancells. In a particular embodiment, the cells used to produce thepackaging cell line are human cells. Suitable cell lines which can beused include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells,COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138cells, MRCS cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells,HeLa cells, W163 cells, 211 cells, and 211A cells. In preferredembodiments, the packaging cells are 293 cells, 293T cells, or A549cells. In another preferred embodiment, the cells are A549 cells.

As used herein, the term “producer cell line” refers to a cell linewhich is capable of producing recombinant retroviral particles,comprising a packaging cell line and a transfer vector constructcomprising a packaging signal. The production of infectious viralparticles and viral stock solutions may be carried out usingconventional techniques. Methods of preparing viral stock solutions areknown in the art and are illustrated by, e.g., Y. Soneoka et al. (1995)Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol.66:5110-5113. Infectious virus particles may be collected from thepackaging cells using conventional techniques. For example, theinfectious particles can be collected by cell lysis, or collection ofthe supernatant of the cell culture, as is known in the art. Optionally,the collected virus particles may be purified if desired. Suitablepurification techniques are well known to those skilled in the art.

The delivery of a gene(s) or other polynucleotide sequence using aretroviral or lentiviral vector by means of viral infection rather thanby transfection is referred to as “transduction.” In one embodiment,retroviral vectors are transduced into a cell through infection andprovirus integration. In certain embodiments, a target cell, e.g., a Tcell, is “transduced” if it comprises a gene or other polynucleotidesequence delivered to the cell by infection using a viral or retroviralvector. In particular embodiments, a transduced cell comprises one ormore genes or other polynucleotide sequences delivered by a retroviralor lentiviral vector in its cellular genome.

In particular embodiments, host cells transduced with viral vector ofthe invention that expresses one or more polypeptides, are administeredto a subject to treat and/or prevent a B cell malignancy. Other methodsrelating to the use of viral vectors in gene therapy, which may beutilized according to certain embodiments of the present invention, canbe found in, e.g., Kay, M. A. (1997) Chest 111(6 Supp.):1385-1425;Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory,Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin.Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther.7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M.(1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994)Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opin.Investig. Drugs 8:2159-2172; Smith-Arica, J. R. and Bartlett, J. S.(2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C. et al. (2000) Nature408:483-8.

G. Genetically Modified Cells

The present invention contemplates, in particular embodiments, cellsgenetically modified to express the CARs contemplated herein, for use inthe treatment of B cell related conditions. As used herein, the term“genetically engineered” or “genetically modified” refers to theaddition of extra genetic material in the form of DNA or RNA into thetotal genetic material in a cell. The terms, “genetically modifiedcells,” “modified cells,” and, “redirected cells,” are usedinterchangeably. As used herein, the term “gene therapy” refers to theintroduction of extra genetic material in the form of DNA or RNA intothe total genetic material in a cell that restores, corrects, ormodifies expression of a gene, or for the purpose of expressing atherapeutic polypeptide, e.g., a CAR.

In particular embodiments, the CARs contemplated herein are introducedand expressed in immune effector cells so as to redirect theirspecificity to a target antigen of interest, e.g., a BCMA polypeptide.An “immune effector cell,” is any cell of the immune system that has oneor more effector functions (e.g., cytotoxic cell killing activity,secretion of cytokines, induction of ADCC and/or CDC).

Immune effector cells of the invention can be autologous/autogeneic(“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic orxenogeneic).

“Autologous,” as used herein, refers to cells from the same subject.

“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison.

“Syngeneic,” as used herein, refers to cells of a different subject thatare genetically identical to the cell in comparison.

“Xenogeneic,” as used herein, refers to cells of a different species tothe cell in comparison. In preferred embodiments, the cells of theinvention are allogeneic.

Illustrative immune effector cells used with the CARs contemplatedherein include T lymphocytes. The terms “T cell” or “T lymphocyte” areart-recognized and are intended to include thymocytes, immature Tlymphocytes, mature T lymphocytes, resting T lymphocytes, or activated Tlymphocytes. A T cell can be a T helper (Th) cell, for example a Thelper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a helper Tcell (HTL; CD4⁺ T cell) CD4⁺ T cell, a cytotoxic T cell (CTL; CD8⁺ Tcell), CD4⁺CD8⁺ T cell, CD4⁺CD8⁺ T cell, or any other subset of T cells.Other illustrative populations of T cells suitable for use in particularembodiments include naïve T cells and memory T cells.

As would be understood by the skilled person, other cells may also beused as immune effector cells with the CARs as described herein. Inparticular, immune effector cells also include NK cells, NKT cells,neutrophils, and macrophages. Immune effector cells also includeprogenitors of effector cells wherein such progenitor cells can beinduced to differentiate into an immune effector cells in vivo or invitro. Thus, in particular embodiments, immune effector cell includesprogenitors of immune effectors cells such as hematopoietic stem cells(HSCs) contained within the CD34+ population of cells derived from cordblood, bone marrow or mobilized peripheral blood which uponadministration in a subject differentiate into mature immune effectorcells, or which can be induced in vitro to differentiate into matureimmune effector cells.

As used herein, immune effector cells genetically engineered to containBCMA-specific CAR may be referred to as, “BCMA-specific redirectedimmune effector cells.”

The term, “CD34+ cell,” as used herein refers to a cell expressing theCD34 protein on its cell surface. “CD34,” as used herein refers to acell surface glycoprotein (e.g., sialomucin protein) that often acts asa cell-cell adhesion factor and is involved in T cell entrance intolymph nodes. The CD34+ cell population contains hematopoietic stem cells(HSC), which upon administration to a patient differentiate andcontribute to all hematopoietic lineages, including T cells, NK cells,NKT cells, neutrophils and cells of the monocyte/macrophage lineage.

The present invention provides methods for making the immune effectorcells which express the CAR contemplated herein. In one embodiment, themethod comprises transfecting or transducing immune effector cellsisolated from an individual such that the immune effector cells expressone or more CAR as described herein. In certain embodiments, the immuneeffector cells are isolated from an individual and genetically modifiedwithout further manipulation in vitro. Such cells can then be directlyre-administered into the individual. In further embodiments, the immuneeffector cells are first activated and stimulated to proliferate invitro prior to being genetically modified to express a CAR. In thisregard, the immune effector cells may be cultured before and/or afterbeing genetically modified (i.e., transduced or transfected to express aCAR contemplated herein).

In particular embodiments, prior to in vitro manipulation or geneticmodification of the immune effector cells described herein, the sourceof cells is obtained from a subject. In particular embodiments, theCAR-modified immune effector cells comprise T cells. T cells can beobtained from a number of sources including, but not limited to,peripheral blood mononuclear cells, bone marrow, lymph nodes tissue,cord blood, thymus issue, tissue from a site of infection, ascites,pleural effusion, spleen tissue, and tumors. In certain embodiments, Tcells can be obtained from a unit of blood collected from a subjectusing any number of techniques known to the skilled person, such assedimentation, e.g., FICOLL™ separation. In one embodiment, cells fromthe circulating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocyte, B cells, other nucleated white blood cells, redblood cells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing. Thecells can be washed with PBS or with another suitable solution thatlacks calcium, magnesium, and most, if not all other, divalent cations.As would be appreciated by those of ordinary skill in the art, a washingstep may be accomplished by methods known to those in the art, such asby using a semiautomated flowthrough centrifuge. For example, the Cobe2991 cell processor, the Baxter CytoMate, or the like. After washing,the cells may be resuspended in a variety of biocompatible buffers orother saline solution with or without buffer. In certain embodiments,the undesirable components of the apheresis sample may be removed in thecell directly resuspended culture media.

In certain embodiments, T cells are isolated from peripheral bloodmononuclear cells (PBMCs) by lysing the red blood cells and depletingthe monocytes, for example, by centrifugation through a PERCOLL™gradient. A specific subpopulation of T cells, expressing one or more ofthe following markers: CD3, CD28, CD4, CD8, CD45RA, and CD45RO, can befurther isolated by positive or negative selection techniques. In oneembodiment, a specific subpopulation of T cells, expressing CD3, CD28,CD4, CD8, CD45RA, and CD45RO is further isolated by positive or negativeselection techniques. For example, enrichment of a T cell population bynegative selection can be accomplished with a combination of antibodiesdirected to surface markers unique to the negatively selected cells. Onemethod for use herein is cell sorting and/or selection via negativemagnetic immunoadherence or flow cytometry that uses a cocktail ofmonoclonal antibodies directed to cell surface markers present on thecells negatively selected. For example, to enrich for CD4⁺ cells bynegative selection, a monoclonal antibody cocktail typically includesantibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometryand cell sorting may also be used to isolate cell populations ofinterest for use in the present invention.

PBMC may be directly genetically modified to express CARs using methodscontemplated herein. In certain embodiments, after isolation of PBMC, Tlymphocytes are further isolated and in certain embodiments, bothcytotoxic and helper T lymphocytes can be sorted into naïve, memory, andeffector T cell subpopulations either before or after geneticmodification and/or expansion.

CD8⁺ cells can be obtained by using standard methods. In someembodiments, CD8⁺ cells are further sorted into naive, central memory,and effector cells by identifying cell surface antigens that areassociated with each of those types of CD8⁺ cells.

In certain embodiments, naive CD8⁺ T lymphocytes are characterized bythe expression of phenotypic markers of naive T cells including CD62L,CCR7, CD28, CD3, CD 127, and CD45RA.

In particular embodiments, memory T cells are present in both CD62L⁺ andCD62L⁻ subsets of CD8⁺ peripheral blood lymphocytes. PBMC are sortedinto CD62L⁻ CD8⁺ and CD62L⁺CD8⁺ fractions after staining with anti-CD8and anti-CD62L antibodies. I n some embodiments, the expression ofphenotypic markers of central memory T cells include CD45RO, CD62L,CCR7, CD28, CD3, and CD127 and are negative for granzyme B. In someembodiments, central memory T cells are CD45RO⁺, CD62L⁺, CD8⁺ T cells.

In some embodiments, effector T cells are negative for CD62L, CCR7,CD28, and CD127, and positive for granzyme B and perforin.

In certain embodiments, CD4⁺ T cells are further sorted intosubpopulations. For example, CD4⁺ T helper cells can be sorted intonaive, central memory, and effector cells by identifying cellpopulations that have cell surface antigens. CD4⁺ lymphocytes can beobtained by standard methods. In some embodiments, naïve CD4⁺ Tlymphocytes are CD45RO⁻, CD45RA⁺, CD62L⁺ CD4⁺ T cell. In someembodiments, central memory CD4⁺ cells are CD62L positive and CD45ROpositive. In some embodiments, effector CD4⁺ cells are CD62L and CD45ROnegative.

The immune effector cells, such as T cells, can be genetically modifiedfollowing isolation using known methods, or the immune effector cellscan be activated and expanded (or differentiated in the case ofprogenitors) in vitro prior to being genetically modified. In aparticular embodiment, the immune effector cells, such as T cells, aregenetically modified with the chimeric antigen receptors contemplatedherein (e.g., transduced with a viral vector comprising a nucleic acidencoding a CAR) and then are activated and expanded in vitro. In variousembodiments, T cells can be activated and expanded before or aftergenetic modification to express a CAR, using methods as described, forexample, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964;5,858,358; 6,887,466; 6,905,681; 7, 144,575; 7,067,318; 7, 172,869;7,232,566; 7, 175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; andU.S. Patent Application Publication No. 20060121005.

Generally, the T cells are expanded by contact with a surface havingattached thereto an agent that stimulates a CD3 TCR complex associatedsignal and a ligand that stimulates a co-stimulatory molecule on thesurface of the T cells. T cell populations may be stimulated by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. Co-stimulation of accessory molecules on the surface of Tcells, is also contemplated.

In particular embodiments, PBMCs or isolated T cells are contacted witha stimulatory agent and costimulatory agent, such as anti-CD3 andanti-CD28 antibodies, generally attached to a bead or other surface, ina culture medium with appropriate cytokines, such as IL-2, IL-7, and/orIL-15. To stimulate proliferation of either CD4⁺ T cells or CD8⁺ Tcells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diacione, Besancon,France) can be used as can other methods commonly known in the art (Berget al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp.Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth.227(1-2):53-63, 1999). Anti-CD3 and anti-CD28 antibodies attached to thesame bead serve as a “surrogate” antigen presenting cell (APC). In otherembodiments, the T cells may be activated and stimulated to proliferatewith feeder cells and appropriate antibodies and cytokines using methodssuch as those described in U.S. Pat. Nos. 6,040,177; 5,827,642; andWO2012129514.

In other embodiments, artificial APC (aAPC) made by engineering K562,U937, 721.221, T2, and C1R cells to direct the stable expression andsecretion, of a variety of co-stimulatory molecules and cytokines. In aparticular embodiment K32 or U32 aAPCs are used to direct the display ofone or more antibody-based stimulatory molecules on the AAPC cellsurface. Expression of various combinations of genes on the aAPC enablesthe precise determination of human T-cell activation requirements, suchthat aAPCs can be tailored for the optimal propagation of T-cell subsetswith specific growth requirements and distinct functions. The aAPCssupport ex vivo growth and long-term expansion of functional human CD8 Tcells without requiring the addition of exogenous cytokines, in contrastto the use of natural APCs. Populations of T cells can be expanded byaAPCs expressing a variety of costimulatory molecules including, but notlimited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86.Finally, the aAPCs provide an efficient platform to expand geneticallymodified T cells and to maintain CD28 expression on CD8 T cells. aAPCsprovided in WO 03/057171 and US2003/0147869 are hereby incorporated byreference in their entirety.

In one embodiment, CD34⁺ cells are transduced with a nucleic acidconstruct in accordance with the invention. In certain embodiments, thetransduced CD34⁺ cells differentiate into mature immune effector cellsin vivo following administration into a subject, generally the subjectfrom whom the cells were originally isolated. In another embodiment,CD34⁺ cells may be stimulated in vitro prior to exposure to or afterbeing genetically modified with a CAR as described herein, with one ormore of the following cytokines: Flt-3 ligand (FLT3), stem cell factor(SCF), megakaryocyte growth and differentiation factor (TPO), IL-3 andIL-6 according to the methods described previously (Asheuer et al.,2004; Imren, et al., 2004).

The invention provides a population of modified immune effector cellsfor the treatment of cancer, the modified immune effector cellscomprising a CAR as disclosed herein. For example, a population ofmodified immune effector cells are prepared from peripheral bloodmononuclear cells (PBMCs) obtained from a patient diagnosed with B cellmalignancy described herein (autologous donors). The PBMCs form aheterogeneous population of T lymphocytes that can be CD4+, CD8+, orCD4+ and CD8+.

The PBMCs also can include other cytotoxic lymphocytes such as NK cellsor NKT cells. An expression vector carrying the coding sequence of a CARcontemplated herein can be introduced into a population of human donor Tcells, NK cells or NKT cells. Successfully transduced T cells that carrythe expression vector can be sorted using flow cytometry to isolate CD3positive T cells and then further propagated to increase the number ofthese CAR protein expressing T cells in addition to cell activationusing anti-CD3 antibodies and or anti-CD28 antibodies and IL-2 or anyother methods known in the art as described elsewhere herein. Standardprocedures are used for cryopreservation of T cells expressing the CARprotein T cells for storage and/or preparation for use in a humansubject. In one embodiment, the in vitro transduction, culture and/orexpansion of T cells are performed in the absence of non-human animalderived products such as fetal calf serum and fetal bovine serum. Sincea heterogeneous population of PBMCs is genetically modified, theresultant transduced cells are a heterogeneous population of modifiedcells comprising a BCMA targeting CAR as contemplated herein.

In a further embodiment, a mixture of, e.g., one, two, three, four, fiveor more, different expression vectors can be used in geneticallymodifying a donor population of immune effector cells wherein eachvector encodes a different chimeric antigen receptor protein ascontemplated herein. The resulting modified immune effector cells formsa mixed population of modified cells, with a proportion of the modifiedcells expressing more than one different CAR proteins.

In one embodiment, the invention provides a method of storinggenetically modified murine, human or humanized CAR protein expressingimmune effector cells which target a BCMA protein, comprisingcryopreserving the immune effector cells such that the cells remainviable upon thawing. A fraction of the immune effector cells expressingthe CAR proteins can be cryopreserved by methods known in the art toprovide a permanent source of such cells for the future treatment ofpatients afflicted with the B cell related condition. When needed, thecryopreserved transformed immune effector cells can be thawed, grown andexpanded for more such cells.

As used herein, “cryopreserving,” refers to the preservation of cells bycooling to sub-zero temperatures, such as (typically) 77 K or −196° C.(the boiling point of liquid nitrogen). Cryoprotective agents are oftenused at sub-zero temperatures to prevent the cells being preserved fromdamage due to freezing at low temperatures or warming to roomtemperature. Cryopreservative agents and optimal cooling rates canprotect against cell injury. Cryoprotective agents which can be usedinclude but are not limited to dimethyl sulfoxide (DMSO) (Lovelock andBishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad.Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin,Nature, 1962; 196: 48). The preferred cooling rate is 1° to 3°C./minute. After at least two hours, the T cells have reached atemperature of −80° C. and can be placed directly into liquid nitrogen(−196° C.) for permanent storage such as in a long-term cryogenicstorage vessel.

H. T Cell Manufacturing Methods

The T cells manufactured by the methods contemplated herein provideimproved adoptive immunotherapy compositions. Without wishing to bebound to any particular theory, it is believed that the T cellcompositions manufactured by the methods contemplated herein are imbuedwith superior properties, including increased survival, expansion in therelative absence of differentiation, and persistence in vivo. In oneembodiment, a method of manufacturing T cells comprises contacting thecells with one or more agents that modulate a PI3K cell signalingpathway. In one embodiment, a method of manufacturing T cells comprisescontacting the cells with one or more agents that modulate aPI3K/Akt/mTOR cell signaling pathway. In various embodiments, the Tcells may be obtained from any source and contacted with the agentduring the activation and/or expansion phases of the manufacturingprocess. The resulting T cell compositions are enriched indevelopmentally potent T cells that have the ability to proliferate andexpress one or more of the following biomarkers: CD62L, CCR7, CD28,CD27, CD122, CD127, CD197, and CD38. In one embodiment, populations ofcell comprising T cells, that have been treated with one or more PI3Kinhibitors is enriched for a population of CD8+ T cells co-expressingone or more or, or all of, the following biomarkers: CD62L, CD127,CD197, and CD38.

In one embodiment, modified T cells comprising maintained levels ofproliferation and decreased differentiation are manufactured. In aparticular embodiment, T cells are manufactured by stimulating T cellsto become activated and to proliferate in the presence of one or morestimulatory signals and an agent that is an inhibitor of a PI3K cellsignaling pathway.

The T cells can then be modified to express an anti-BCMA CARs. In oneembodiment, the T cells are modified by transducing the T cells with aviral vector comprising an anti-BCMA CAR contemplated herein. In acertain embodiment, the T cells are modified prior to stimulation andactivation in the presence of an inhibitor of a PI3K cell signalingpathway. In another embodiment, T cells are modified after stimulationand activation in the presence of an inhibitor of a PI3K cell signalingpathway. In a particular embodiment, T cells are modified within 12hours, 24 hours, 36 hours, or 48 hours of stimulation and activation inthe presence of an inhibitor of a PI3K cell signaling pathway.

After T cells are activated, the cells are cultured to proliferate. Tcells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more rounds of expansion.

In various embodiments, T cell compositions are manufactured in thepresence of one or more inhibitors of the PI3K pathway. The inhibitorsmay target one or more activities in the pathway or a single activity.Without wishing to be bound to any particular theory, it is contemplatedthat treatment or contacting T cells with one or more inhibitors of thePI3K pathway during the stimulation, activation, and/or expansion phasesof the manufacturing process preferentially increases young T cells,thereby producing superior therapeutic T cell compositions.

In a particular embodiment, a method for increasing the proliferation ofT cells expressing an engineered T cell receptor is provided. Suchmethods may comprise, for example, harvesting a source of T cells from asubject, stimulating and activating the T cells in the presence of oneor more inhibitors of the PI3K pathway, modification of the T cells toexpress an anti-BCMA CAR, e.g., anti-BCMA02 CAR, and expanding the Tcells in culture.

In a certain embodiment, a method for producing populations of T cellsenriched for expression of one or more of the following biomarkers:CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In oneembodiment, young T cells comprise one or more of, or all of thefollowing biological markers: CD62L, CD127, CD197, and CD38. In oneembodiment, the young T cells lack expression of CD57, CD244, CD160,PD-1, CTLA4, TIM3, and LAG3 are provided. As discussed elsewhere herein,the expression levels young T cell biomarkers is relative to theexpression levels of such markers in more differentiated T cells orimmune effector cell populations.

In one embodiment, peripheral blood mononuclear cells (PBMCs) are usedas the source of T cells in the T cell manufacturing methodscontemplated herein. PBMCs form a heterogeneous population of Tlymphocytes that can be CD4⁺, CD8⁺, or CD4⁺ and CD8⁺ and can includeother mononuclear cells such as monocytes, B cells, NK cells and NKTcells. An expression vector comprising a polynucleotide encoding anengineered TCR or CAR contemplated herein can be introduced into apopulation of human donor T cells, NK cells or NKT cells. Successfullytransduced T cells that carry the expression vector can be sorted usingflow cytometry to isolate CD3 positive T cells and then furtherpropagated to increase the number of the modified T cells in addition tocell activation using anti-CD3 antibodies and or anti-CD28 antibodiesand IL-2, IL-7, and/or IL-15 or any other methods known in the art asdescribed elsewhere herein.

Manufacturing methods contemplated herein may further comprisecryopreservation of modified T cells for storage and/or preparation foruse in a human subject. T cells are cryopreserved such that the cellsremain viable upon thawing. When needed, the cryopreserved transformedimmune effector cells can be thawed, grown and expanded for more suchcells. As used herein, “cryopreserving,” refers to the preservation ofcells by cooling to sub-zero temperatures, such as (typically) 77 K or−196° C. (the boiling point of liquid nitrogen). Cryoprotective agentsare often used at sub-zero temperatures to prevent the cells beingpreserved from damage due to freezing at low temperatures or warming toroom temperature. Cryopreservative agents and optimal cooling rates canprotect against cell injury. Cryoprotective agents which can be usedinclude but are not limited to dimethyl sulfoxide (DMSO) (Lovelock andBishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190:1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad.Sci., 1960; 85: 576), and polyethylene glycol (Sloviter and Ravdin,Nature, 1962; 196: 48). The preferred cooling rate is 1° to 3°C./minute. After at least two hours, the T cells have reached atemperature of −80° C. and can be placed directly into liquid nitrogen(−196° C.) for permanent storage such as in a long-term cryogenicstorage vessel.

1. T Cells

The present invention contemplates the manufacture of improved CART cellcompositions. T cells used for CAR T cell production may beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic). In preferred embodiments, the Tcells are obtained from a mammalian subject. In a more preferredembodiment, the T cells are obtained from a primate subject. In the mostpreferred embodiment, the T cells are obtained from a human subject.

T cells can be obtained from a number of sources including, but notlimited to, peripheral blood mononuclear cells, bone marrow, lymph nodestissue, cord blood, thymus issue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled person,such as sedimentation, e.g., FICOLL™ separation. In one embodiment,cells from the circulating blood of an individual are obtained byapheresis. The apheresis product typically contains lymphocytes,including T cells, monocytes, granulocytes, B cells, other nucleatedwhite blood cells, red blood cells, and platelets. In one embodiment,the cells collected by apheresis may be washed to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing. The cells can be washed with PBS or with anothersuitable solution that lacks calcium, magnesium, and most, if not allother, divalent cations. As would be appreciated by those of ordinaryskill in the art, a washing step may be accomplished by methods known tothose in the art, such as by using a semiautomated flowthroughcentrifuge. For example, the Cobe 2991 cell processor, the BaxterCytoMate, or the like. After washing, the cells may be resuspended in avariety of biocompatible buffers or other saline solution with orwithout buffer. In certain embodiments, the undesirable components ofthe apheresis sample may be removed in the cell directly resuspendedculture media.

In particular embodiments, a population of cells comprising T cells,e.g., PBMCs, is used in the manufacturing methods contemplated herein.In other embodiments, an isolated or purified population of T cells isused in the manufacturing methods contemplated herein. Cells can beisolated from peripheral blood mononuclear cells (PBMCs) by lysing thered blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. In some embodiments, afterisolation of PBMC, both cytotoxic and helper T lymphocytes can be sortedinto naïve, memory, and effector T cell subpopulations either before orafter activation, expansion, and/or genetic modification.

A specific subpopulation of T cells, expressing one or more of thefollowing markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, andHLA-DR can be further isolated by positive or negative selectiontechniques. In one embodiment, a specific subpopulation of T cells,expressing one or more of the markers selected from the group consistingof i) CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or ii) CD38 orCD62L, CD127, CD197, and CD3ε, is further isolated by positive ornegative selection techniques. In various embodiments, the manufacturedT cell compositions do not express or do not substantially express oneor more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3,and LAG3.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD62L, CD127, CD197, and CD38 is increasedat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 25 fold, or more compared to apopulation of T cells activated and expanded without a PI3K inhibitor.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, andLAG3 is decreased at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, ormore compared to a population of T cells activated and expanded with aPI3K inhibitor.

In one embodiment, the manufacturing methods contemplated hereinincrease the number CAR T cells comprising one or more markers of naïveor developmentally potent T cells. Without wishing to be bound to anyparticular theory, the present inventors believe that treating apopulation of cells comprising T cells with one or more PI3K inhibitorsresults in an increase an expansion of developmentally potent T cellsand provides a more robust and efficacious adoptive CAR T cellimmunotherapy compared to existing CAR T cell therapies.

Illustrative examples of markers of naïve or developmentally potent Tcells increased in T cells manufactured using the methods contemplatedherein include, but are not limited to CD62L, CD127, CD197, and CD38. Inparticular embodiments, naïve T cells do not express do not express ordo not substantially express one or more of the following markers: CD57,CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3, and LAG3.

With respect to T cells, the T cell populations resulting from thevarious expansion methodologies contemplated herein may have a varietyof specific phenotypic properties, depending on the conditions employed.In various embodiments, expanded T cell populations comprise one or moreof the following phenotypic markers: CD62L, CD127, CD197, CD3ε, andHLA-DR.

In one embodiment, such phenotypic markers include enhanced expressionof one or more of, or all of CD62L, CD127, CD197, and CD38. Inparticular embodiments, CD8⁺ T lymphocytes characterized by theexpression of phenotypic markers of naive T cells including CD62L,CD127, CD197, and CD38 are expanded.

In particular embodiments, T cells characterized by the expression ofphenotypic markers of central memory T cells including CD45RO, CD62L,CD127, CD197, and CD38 and negative for granzyme B are expanded. In someembodiments, the central memory T cells are CD45RO⁺, CD62L⁺, CD8⁺ Tcells.

In certain embodiments, CD4⁺ T lymphocytes characterized by theexpression of phenotypic markers of naïve CD4⁺ cells including CD62L andnegative for expression of CD45RA and/or CD45RO are expanded. In someembodiments, CD4⁺ cells characterized by the expression of phenotypicmarkers of central memory CD4⁺ cells including CD62L and CD45ROpositive. In some embodiments, effector CD4⁺ cells are CD62L positiveand CD45RO negative.

In certain embodiments, the T cells are isolated from an individual andactivated and stimulated to proliferate in vitro prior to beinggenetically modified to express an anti-BCMA CAR. In this regard, the Tcells may be cultured before and/or after being genetically modified(i.e., transduced or transfected to express an anti-BCMA CARcontemplated herein).

2. Activation and Expansion

In order to achieve sufficient therapeutic doses of T cell compositions,T cells are often subject to one or more rounds of stimulation,activation and/or expansion. T cells can be activated and expandedgenerally using methods as described, for example, in Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which isincorporated herein by reference in its entirety. T cells modified toexpress an anti-BCMA CAR can be activated and expanded before and/orafter the T cells are modified. In addition, T cells may be contactedwith one or more agents that modulate the PI3K cell signaling pathwaybefore, during, and/or after activation and/or expansion. In oneembodiment, T cells manufactured by the methods contemplated hereinundergo one, two, three, four, or five or more rounds of activation andexpansion, each of which may include one or more agents that modulatethe PI3K cell signaling pathway.

In one embodiment, a costimulatory ligand is presented on an antigenpresenting cell (e.g., an aAPC, dendritic cell, B cell, and the like)that specifically binds a cognate costimulatory molecule on a T cell,thereby providing a signal which, in addition to the primary signalprovided by, for instance, binding of a TCR/CD3 complex, mediates adesired T cell response. Suitable costimulatory ligands include, but arenot limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, ILT3, ILT4, an agonist or antibody that bindsToll ligand receptor, and a ligand that specifically binds with B7-H3.

In a particular embodiment, a costimulatory ligand comprises an antibodyor antigen binding fragment thereof that specifically binds to acostimulatory molecule present on a T cell, including but not limitedto, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, 1COS, lymphocytefunction-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and aligand that specifically binds with CD83.

Suitable costimulatory ligands further include target antigens, whichmay be provided in soluble form or expressed on APCs or aAPCs that bindengineered TCRs or CARs expressed on modified T cells.

In various embodiments, a method for manufacturing T cells contemplatedherein comprises activating a population of cells comprising T cells andexpanding the population of T cells. T cell activation can beaccomplished by providing a primary stimulation signal through the Tcell TCR/CD3 complex or via stimulation of the CD2 surface protein andby providing a secondary costimulation signal through an accessorymolecule, e.g, CD28.

The TCR/CD3 complex may be stimulated by contacting the T cell with asuitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonalantibody. Illustrative examples of CD3 antibodies include, but are notlimited to, OKT3, G19-4, BC3, and 64.1.

In another embodiment, a CD2 binding agent may be used to provide aprimary stimulation signal to the T cells. Illustrative examples of CD2binding agents include, but are not limited to, CD2 ligands and anti-CD2antibodies, e.g., the T11.3 antibody in combination with the T11.1 orT11.2 antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6antibody (which recognizes the same epitope as TI 1.1) in combinationwith the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol.137:1097-1100). Other antibodies which bind to the same epitopes as anyof the above described antibodies can also be used. Additionalantibodies, or combinations of antibodies, can be prepared andidentified by standard techniques as disclosed elsewhere herein.

In addition to the primary stimulation signal provided through theTCR/CD3 complex, or via CD2, induction of T cell responses requires asecond, costimulatory signal. In particular embodiments, a CD28 bindingagent can be used to provide a costimulatory signal. Illustrativeexamples of CD28 binding agents include but are not limited to: naturalCD 28 ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7family of proteins, such as B7-1(CD80) and B7-2 (CD86); and anti-CD28monoclonal antibody or fragment thereof capable of crosslinking the CD28molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8,248.23.2, and EX5.3D10.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are coupled tothe same surface.

In certain embodiments, binding agents that provide stimulatory andcostimulatory signals are localized on the surface of a cell. This canbe accomplished by transfecting or transducing a cell with a nucleicacid encoding the binding agent in a form suitable for its expression onthe cell surface or alternatively by coupling a binding agent to thecell surface.

In another embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are displayed onantigen presenting cells.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex or CD2, and the costimulatory molecule are provided onseparate surfaces.

In a certain embodiment, one of the binding agents that providestimulatory and costimulatory signals is soluble (provided in solution)and the other agent(s) is provided on one or more surfaces.

In a particular embodiment, the binding agents that provide stimulatoryand costimulatory signals are both provided in a soluble form (providedin solution).

In various embodiments, the methods for manufacturing T cellscontemplated herein comprise activating T cells with anti-CD3 andanti-CD28 antibodies.

T cell compositions manufactured by the methods contemplated hereincomprise T cells activated and/or expanded in the presence of one ormore agents that inhibit a PI3K cell signaling pathway. T cells modifiedto express an anti-BCMA CAR can be activated and expanded before and/orafter the T cells are modified. In particular embodiments, a populationof T cells is activated, modified to express an anti-BCMA CAR, and thencultured for expansion.

In one embodiment, T cells manufactured by the methods contemplatedherein comprise an increased number of T cells expressing markersindicative of high proliferative potential and the ability to self-renewbut that do not express or express substantially undetectable markers ofT cell differentiation. These T cells may be repeatedly activated andexpanded in a robust fashion and thereby provide an improved therapeuticT cell composition.

In one embodiment, a population of T cells activated and expanded in thepresence of one or more agents that inhibit a PI3K cell signalingpathway is expanded at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, atleast 50 fold, at least 100 fold, at least 250 fold, at least 500 fold,at least 1000 fold, or more compared to a population of T cellsactivated and expanded without a PI3K inhibitor.

In one embodiment, a population of T cells characterized by theexpression of markers young T cells are activated and expanded in thepresence of one or more agents that inhibit a PI3K cell signalingpathway is expanded at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, atleast 50 fold, at least 100 fold, at least 250 fold, at least 500 fold,at least 1000 fold, or more compared the population of T cells activatedand expanded without a PI3K inhibitor.

In one embodiment, expanding T cells activated by the methodscontemplated herein further comprises culturing a population of cellscomprising T cells for several hours (about 3 hours) to about 7 days toabout 28 days or any hourly integer value in between. In anotherembodiment, the T cell composition may be cultured for 14 days. In aparticular embodiment, T cells are cultured for about 21 days. Inanother embodiment, the T cell compositions are cultured for about 2-3days. Several cycles of stimulation/activation/expansion may also bedesired such that culture time of T cells can be 60 days or more.

In particular embodiments, conditions appropriate for T cell cultureinclude an appropriate media (e.g., Minimal Essential Media or RPMIMedia 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary forproliferation and viability including, but not limited to serum (e.g.,fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ,IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or anyother additives suitable for the growth of cells known to the skilledartisan.

Further illustrative examples of cell culture media include, but are notlimited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 5,and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.

Illustrative examples of other additives for T cell expansion include,but are not limited to, surfactant, piasmanate, pH buffers such asHEPES, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanol

Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C02).

In particular embodiments, PBMCs or isolated T cells are contacted witha stimulatory agent and costimulatory agent, such as anti-CD3 andanti-CD28 antibodies, generally attached to a bead or other surface, ina culture medium with appropriate cytokines, such as IL-2, IL-7, and/orIL-15.

In other embodiments, artificial APC (aAPC) made by engineering K562,U937, 721.221, T2, and C1R cells to direct the stable expression andsecretion, of a variety of costimulatory molecules and cytokines. In aparticular embodiment K32 or U32 aAPCs are used to direct the display ofone or more antibody-based stimulatory molecules on the AAPC cellsurface. Populations of T cells can be expanded by aAPCs expressing avariety of costimulatory molecules including, but not limited to, CD137L(4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Finally, the aAPCsprovide an efficient platform to expand genetically modified T cells andto maintain CD28 expression on CD8 T cells. aAPCs provided in WO03/057171 and US2003/0147869 are hereby incorporated by reference intheir entirety.

3. Agents

In various embodiments, a method for manufacturing T cells is providedthat expands undifferentiated or developmentally potent T cellscomprising contacting T cells with an agent that modulates a PI3Kpathway in the cells. In various embodiments, a method for manufacturingT cells is provided that expands undifferentiated or developmentallypotent T cells comprising contacting T cells with an agent thatmodulates a PI3K/AKT/mTOR pathway in the cells. The cells may becontacted prior to, during, and/or after activation and expansion. The Tcell compositions retain sufficient T cell potency such that they mayundergo multiple rounds of expansion without a substantial increase indifferentiation.

As used herein, the terms “modulate,” “modulator,” or “modulatory agent”or comparable term refer to an agent's ability to elicit a change in acell signaling pathway. A modulator may increase or decrease an amount,activity of a pathway component or increase or decrease a desired effector output of a cell signaling pathway. In one embodiment, the modulatoris an inhibitor. In another embodiment, the modulator is an activator.

An “agent” refers to a compound, small molecule, e.g., small organicmolecule, nucleic acid, polypeptide, or a fragment, isoform, variant,analog, or derivative thereof used in the modulation of a PI3K/AKT/mTORpathway.

A “small molecule” refers to a composition that has a molecular weightof less than about 5 kD, less than about 4 kD, less than about 3 kD,less than about 2 kD, less than about 1 kD, or less than about 0.5 kD.Small molecules may comprise nucleic acids, peptides, polypeptides,peptidomimetics, peptoids, carbohydrates, lipids, components thereof orother organic or inorganic molecules. Libraries of chemical and/orbiological mixtures, such as fungal, bacterial, or algal extracts, areknown in the art and can be screened with any of the assays of theinvention. Examples of methods for the synthesis of molecular librariescan be found in: (Carell et al., 1994a; Carell et al., 1994b; Cho etal., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al.,1994).

An “analog” refers to a small organic compound, a nucleotide, a protein,or a polypeptide that possesses similar or identical activity orfunction(s) as the compound, nucleotide, protein or polypeptide orcompound having the desired activity of the present invention, but neednot necessarily comprise a sequence or structure that is similar oridentical to the sequence or structure of the preferred embodiment.

A “derivative” refers to either a compound, a protein or polypeptidethat comprises an amino acid sequence of a parent protein or polypeptidethat has been altered by the introduction of amino acid residuesubstitutions, deletions or additions, or a nucleic acid or nucleotidethat has been modified by either introduction of nucleotidesubstitutions or deletions, additions or mutations. The derivativenucleic acid, nucleotide, protein or polypeptide possesses a similar oridentical function as the parent polypeptide.

In various embodiments, the agent that modulates a PI3K pathwayactivates a component of the pathway. An “activator,” or “agonist”refers to an agent that promotes, increases, or induces one or moreactivities of a molecule in a PI3K/AKT/mTOR pathway including, withoutlimitation, a molecule that inhibits one or more activities of a PI3K.

In various embodiments, the agent that modulates a PI3K pathway inhibitsa component of the pathway. An “inhibitor” or “antagonist” refers to anagent that inhibits, decreases, or reduces one or more activities of amolecule in a PI3K pathway including, without limitation, a PI3K. In oneembodiment, the inhibitor is a dual molecule inhibitor. In particularembodiment, the inhibitor may inhibit a class of molecules have the sameor substantially similar activities (a pan-inhibitor) or mayspecifically inhibit a molecule's activity (a selective or specificinhibitor). Inhibition may also be irreversible or reversible.

In one embodiment, the inhibitor has an IC50 of at least 1 nM, at least2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, atleast 200 nM, at least 500 nM, at least 1 μM, at least 10 μM, at least50 μM, or at least 100 μM. IC50 determinations can be accomplished usingany conventional techniques known in the art. For example, an IC50 canbe determined by measuring the activity of a given enzyme in thepresence of a range of concentrations of the inhibitor under study. Theexperimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the “IC50” value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity.

In various embodiments, T cells are contacted or treated or culturedwith one or more modulators of a PI3K pathway at a concentration of atleast 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 μM, atleast 10 μM, at least 50 μM, at least 100 μM, or at least 1 M.

In particular embodiments, T cells may be contacted or treated orcultured with one or more modulators of a PI3K pathway for at least 12hours, 18 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks,at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more rounds of expansion.

a. PI3K/Akt/mTOR Pathway

The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycinpathway serves as a conduit to integrate growth factor signaling withcellular proliferation, differentiation, metabolism, and survival. PI3Ksare a family of highly conserved intracellular lipid kinases. Class IAPI3Ks are activated by growth factor receptor tyrosine kinases (RTKs),either directly or through interaction with the insulin receptorsubstrate family of adaptor molecules. This activity results in theproduction of phosphatidyl-inositol-3,4,5-trisphospate (PIP3) aregulator of the serine/threonine kinase Akt. mTOR acts through thecanonical PI3K pathway via 2 distinct complexes, each characterized bydifferent binding partners that confer distinct activities. mTORC1 (mTORin complex with PRAS40, raptor, and mLST8/GbL) acts as a downstreameffector of PI3K/Akt signaling, linking growth factor signals withprotein translation, cell growth, proliferation, and survival. mTORC2(mTOR in complex with rictor, mSIN1, protor, and mLST8) acts as anupstream activator of Akt.

Upon growth factor receptor-mediated activation of PI3K, Akt isrecruited to the membrane through the interaction of its pleckstrinhomology domain with PIP3, thus exposing its activation loop andenabling phosphorylation at threonine 308 (Thr308) by the constitutivelyactive phosphoinositide-dependent protein kinase 1 (PDK1). For maximalactivation, Akt is also phosphorylated by mTORC2, at serine 473 (Ser473)of its C-terminal hydrophobic motif. DNA-PK and HSP have also been shownto be important in the regulation of Akt activity. Akt activates mTORC1through inhibitory phosphorylation of TSC2, which along with TSC1,negatively regulates mTORC1 by inhibiting the Rheb GTPase, a positiveregulator of mTORC1. mTORC1 has 2 well-defined substrates, p70S6K(referred to hereafter as S6K1) and 4E-BP1, both of which criticallyregulate protein synthesis. Thus, mTORC1 is an important downstreameffector of PI3K, linking growth factor signaling with proteintranslation and cellular proliferation.

b. PI3K Inhibitors

As used herein, the term “PI3K inhibitor” refers to a nucleic acid,peptide, compound, or small organic molecule that binds to and inhibitsat least one activity of PI3K. The PI3K proteins can be divided intothree classes, class 1 PI3Ks, class 2 PI3Ks, and class 3 PI3Ks. Class 1PI3Ks exist as heterodimers consisting of one of four p110 catalyticsubunits (p110α, p110β, p110δ, and p110γ) and one of two families ofregulatory subunits. A PI3K inhibitor of the present inventionpreferably targets the class 1 PI3K inhibitors. In one embodiment, aPI3K inhibitor will display selectivity for one or more isoforms of theclass 1 PI3K inhibitors (i.e., selectivity for p110α, p110β, p110δ, andp110γ or one or more of p110α, p110β, p110δ, and p110γ). In anotheraspect, a PI3K inhibitor will not display isoform selectivity and beconsidered a “pan-PI3K inhibitor.” In one embodiment, a PI3K inhibitorwill compete for binding with ATP to the PI3K catalytic domain.

In certain embodiments, a PI3K inhibitor can, for example, target PI3Kas well as additional proteins in the PI3K-AKT-mTOR pathway. Inparticular embodiments, a PI3K inhibitor that targets both mTOR and PI3Kcan be referred to as either an mTOR inhibitor or a PI3K inhibitor. API3K inhibitor that only targets PI3K can be referred to as a selectivePI3K inhibitor. In one embodiment, a selective PI3K inhibitor can beunderstood to refer to an agent that exhibits a 50% inhibitoryconcentration with respect to PI3K that is at least 10-fold, at least20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least1000-fold, or more, lower than the inhibitor's IC50 with respect to mTORand/or other proteins in the pathway.

In a particular embodiment, exemplary PI3K inhibitors inhibit PI3K withan IC50 (concentration that inhibits 50% of the activity) of about 200nM or less, preferably about 100 nm or less, even more preferably about60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM,50 μM, 25 μM, 10 μM, 1 μM, or less. In one embodiment, a PI3K inhibitorinhibits PI3K with an IC50 from about 2 nM to about 100 nm, morepreferably from about 2 nM to about 50 nM, even more preferably fromabout 2 nM to about 15 nM.

Illustrative examples of PI3K inhibitors suitable for use in the T cellmanufacturing methods contemplated herein include, but are not limitedto, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3Kinhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline), and PX-866(class 1 PI3K inhibitor; p110α, p110β, and p110γ isoforms, Oncothyreon).

Other illustrative examples of selective PI3K inhibitors include, butare not limited to BYL719, GSK2636771, TGX-221, AS25242, CAL-101,ZSTK474, and IPI-145.

Further illustrative examples of pan-PI3K inhibitors include, but arenot limited to BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

c. AKT Inhibitors

As used herein, the term “AKT inhibitor” refers to a nucleic acid,peptide, compound, or small organic molecule that inhibits at least oneactivity of AKT. AKT inhibitors can be grouped into several classes,including lipid-based inhibitors (e.g., inhibitors that target thepleckstrin homology domain of AKT which prevents AKT from localizing toplasma membranes), ATP-competitive inhibitors, and allostericinhibitors. In one embodiment, AKT inhibitors act by binding to the AKTcatalytic site. In a particular embodiment, Akt inhibitors act byinhibiting phosphorylation of downstream AKT targets such as mTOR. Inanother embodiment, AKT activity is inhibited by inhibiting the inputsignals to activate Akt by inhibiting, for example, DNA-PK activation ofAKT, PDK-1 activation of AKT, and/or mTORC2 activation of Akt.

AKT inhibitors can target all three AKT isoforms, AKT1, AKT2, AKT3 ormay be isoform selective and target only one or two of the AKT isoforms.In one embodiment, an AKT inhibitor can target AKT as well as additionalproteins in the PI3K-AKT-mTOR pathway. An AKT inhibitor that onlytargets AKT can be referred to as a selective AKT inhibitor. In oneembodiment, a selective AKT inhibitor can be understood to refer to anagent that exhibits a 50% inhibitory concentration with respect to AKTthat is at least 10-fold, at least 20-fold, at least 30-fold, at least50-fold, at least 100-fold, at least 1000-fold, or more lower than theinhibitor's IC50 with respect to other proteins in the pathway.

In a particular embodiment, exemplary AKT inhibitors inhibit AKT with anIC50 (concentration that inhibits 50% of the activity) of about 200 nMor less, preferably about 100 nm or less, even more preferably about 60nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50μM, 25 μM, 10 μM, 1 μM, or less. In one embodiment, an AKT inhibits AKTwith an IC50 from about 2 nM to about 100 nm, more preferably from about2 nM to about 50 nM, even more preferably from about 2 nM to about 15nM.

Illustrative examples of AKT inhibitors for use in combination withauristatin based antibody-drug conjugates include, for example,perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418(Exelixis), GSK690693, GDC-0068, and PX316 (PROLX Pharmaceuticals).

An illustrative, non-limiting example of a selective Akt1 inhibitor isA-674563.

An illustrative, non-limiting example of a selective Akt2 inhibitor isCCT128930.

In particular embodiments, the Akt inhibitor DNA-PK activation of Akt,PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation ofAkt.

Illustrative examples of DNA-PK inhibitors include, but are not limitedto, NU7441, PI-103, NU7026, PIK-75, and PP-121.

d. mTOR Inhibitors

The terms “mTOR inhibitor” or “agent that inhibits mTOR” refers to anucleic acid, peptide, compound, or small organic molecule that inhibitsat least one activity of an mTOR protein, such as, for example, theserine/threonine protein kinase activity on at least one of itssubstrates (e.g., p70S6 kinase 1, 4E-BP1, AKT/PKB and eEF2). mTORinhibitors are able to bind directly to and inhibit mTORC1, mTORC2 orboth mTORC1 and mTORC2.

Inhibition of mTORC1 and/or mTORC2 activity can be determined by areduction in signal transduction of the PI3K/Akt/mTOR pathway. A widevariety of readouts can be utilized to establish a reduction of theoutput of such signaling pathway. Some non-limiting exemplary readoutsinclude (1) a decrease in phosphorylation of Akt at residues, includingbut not limited to 5473 and T308; (2) a decrease in activation of Akt asevidenced, for example, by a reduction of phosphorylation of Aktsubstrates including but not limited to Fox01/O3a T24/32, GSK3α/β;S21/9, and TSC2 T1462; (3) a decrease in phosphorylation of signalingmolecules downstream of mTOR, including but not limited to ribosomal S6S240/244, 70S6K T389, and 4EBP1 T37/46; and (4) inhibition ofproliferation of cancerous cells.

In one embodiment, the mTOR inhibitors are active site inhibitors. Theseare mTOR inhibitors that bind to the ATP binding site (also referred toas ATP binding pocket) of mTOR and inhibit the catalytic activity ofboth mTORC1 and mTORC2. One class of active site inhibitors suitable foruse in the T cell manufacturing methods contemplated herein are dualspecificity inhibitors that target and directly inhibit both PI3K andmTOR. Dual specificity inhibitors bind to both the ATP binding site ofmTOR and PI3K. Illustrative examples of such inhibitors include, but arenot limited to: imidazoquinazolines, wortmannin, LY294002, PI-103(Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765(Exelixis) and NVP-BEZ235 (Novartis).

Another class of mTOR active site inhibitors suitable for use in themethods contemplated herein selectively inhibit mTORC1 and mTORC2activity relative to one or more type I phophatidylinositol 3-kinases,e.g., PI3 kinase α, β, γ, or δ. These active site inhibitors bind to theactive site of mTOR but not PI3K. Illustrative examples of suchinhibitors include, but are not limited to: pyrazolopyrimidines, Torinl(Guertin and Sabatini), PP242(2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol),PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth), andAZD8055 (Liu et al., Nature Review, 8, 627-644, 2009). I

In one embodiment, a selective mTOR inhibitor refers to an agent thatexhibits a 50% inhibitory concentration (IC50) with respect to mTORC1and/or mTORC2, that is at least 10-fold, at least 20-fold, at least50-fold, at least 100-fold, at least 1000-fold, or more, lower than theinhibitor's IC50 with respect to one, two, three, or more type IPI3-kinases or to all of the type I PI3-kinases.

Another class of mTOR inhibitors for use in the present invention arereferred to herein as “rapalogs”. As used herein the term “rapalogs”refers to compounds that specifically bind to the mTOR FRB domain (FKBPrapamycin binding domain), are structurally related to rapamycin, andretain the mTOR inhibiting properties. The term rapalogs excludesrapamycin. Rapalogs include esters, ethers, oximes, hydrazones, andhydroxylamines of rapamycin, as well as compounds in which functionalgroups on the rapamycin core structure have been modified, for example,by reduction or oxidation. Pharmaceutically acceptable salts of suchcompounds are also considered to be rapamycin derivatives. Illustrativeexamples of rapalogs suitable for use in the methods contemplated hereininclude, without limitation, temsirolimus (CC1779), everolimus (RAD001),deforolimus (AP23573), AZD8055 (AstraZeneca), and OSI-027 (OSI).

In one embodiment, the agent is the mTOR inhibitor rapamycin(sirolimus).

In a particular embodiment, exemplary mTOR inhibitors for use in thepresent invention inhibit either mTORC1, mTORC2 or both mTORC1 andmTORC2 with an IC50 (concentration that inhibits 50% of the activity) ofabout 200 nM or less, preferably about 100 nm or less, even morepreferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM,about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM, or less. In one aspect, amTOR inhibitor for use in the present invention inhibits either mTORC1,mTORC2 or both mTORC1 and mTORC2 with an IC50 from about 2 nM to about100 nm, more preferably from about 2 nM to about 50 nM, even morepreferably from about 2 nM to about 15 nM.

In one embodiment, exemplary mTOR inhibitors inhibit either PI3K andmTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC50(concentration that inhibits 50% of the activity) of about 200 nM orless, preferably about 100 nm or less, even more preferably about 60 nMor less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50μM, 25 μM, 10 μM, 1 μM, or less. In one aspect, a mTOR inhibitor for usein the present invention inhibits PI3K and mTORC1 or mTORC2 or bothmTORC1 and mTORC2 and PI3K with an IC50 from about 2 nM to about 100 nm,more preferably from about 2 nM to about 50 nM, even more preferablyfrom about 2 nM to about 15 nM.

Further illustrative examples of mTOR inhibitors suitable for use inparticular embodiments contemplated herein include, but are not limitedto AZD8055, INK128, rapamycin, PF-04691502, and everolimus.

mTOR has been shown to demonstrate a robust and specific catalyticactivity toward the physiological substrate proteins, p70 S6 ribosomalprotein kinase I (p70S6K1) and eIF4E binding protein 1 (4EBP1) asmeasured by phosphor-specific antibodies in Western blotting.

In one embodiment, the inhibitor of the PI3K/AKT/mTOR pathway is a s6kinase inhibitor selected from the group consisting of: BI-D1870, H89,PF-4708671, FMK, and AT7867.

I. Compositions and Formulations

The compositions contemplated herein may comprise one or morepolypeptides, polynucleotides, vectors comprising same, geneticallymodified immune effector cells, etc., as contemplated herein.Compositions include, but are not limited to pharmaceuticalcompositions. A “pharmaceutical composition” refers to a compositionformulated in pharmaceutically-acceptable or physiologically-acceptablesolutions for administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy. It will alsobe understood that, if desired, the compositions of the invention may beadministered in combination with other agents as well, such as, e.g.,cytokines, growth factors, hormones, small molecules, chemotherapeutics,pro-drugs, drugs, antibodies, or other various pharmaceutically-activeagents. There is virtually no limit to other components that may also beincluded in the compositions, provided that the additional agents do notadversely affect the ability of the composition to deliver the intendedtherapy.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations.

In particular embodiments, compositions of the present inventioncomprise an amount of CAR-expressing immune effector cells contemplatedherein. As used herein, the term “amount” refers to “an amounteffective” or “an effective amount” of a genetically modifiedtherapeutic cell, e.g., T cell, to achieve a beneficial or desiredprophylactic or therapeutic result, including clinical results.

A “prophylactically effective amount” refers to an amount of agenetically modified therapeutic cell effective to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

A “therapeutically effective amount” of a genetically modifiedtherapeutic cell may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thestem and progenitor cells to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the virus or transduced therapeuticcells are outweighed by the therapeutically beneficial effects. The term“therapeutically effective amount” includes an amount that is effectiveto “treat” a subject (e.g., a patient). When a therapeutic amount isindicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10² to 10¹⁰ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. The number ofcells will depend upon the ultimate use for which the composition isintended as will the type of cells included therein. For uses providedherein, the cells are generally in a volume of a liter or less, can be500 mL or less, even 250 mL or 100 mL or less. Hence the density of thedesired cells is typically greater than 10⁶ cells/ml and generally isgreater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. Theclinically relevant number of immune cells can be apportioned intomultiple infusions that cumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some aspects of the presentinvention, particularly since all the infused cells will be redirectedto a particular target antigen (e.g., κ or λ light chain), lower numbersof cells, in the range of 10⁶/kilogram (10⁶-10¹¹ per patient) may beadministered. CAR expressing cell compositions may be administeredmultiple times at dosages within these ranges. The cells may beallogeneic, syngeneic, xenogeneic, or autologous to the patientundergoing therapy. If desired, the treatment may also includeadministration of mitogens (e.g., PHA) or lymphokines, cytokines, and/orchemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta,GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein toenhance induction of the immune response.

Generally, compositions comprising the cells activated and expanded asdescribed herein may be utilized in the treatment and prevention ofdiseases that arise in individuals who are immunocompromised. Inparticular, compositions comprising the CAR-modified T cellscontemplated herein are used in the treatment of B cell malignancies.The CAR-modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withcarriers, diluents, excipients, and/or with other components such asIL-2 or other cytokines or cell populations. In particular embodiments,pharmaceutical compositions contemplated herein comprise an amount ofgenetically modified T cells, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients.

Pharmaceutical compositions of the present invention comprising aCAR-expressing immune effector cell population, such as T cells, maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, mannose, sucrose ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions ofthe present invention are preferably formulated for parenteraladministration, e.g., intravascular (intravenous or intraarterial),intraperitoneal or intramuscular administration.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. An injectablepharmaceutical composition is preferably sterile.

In a particular embodiment, compositions contemplated herein comprise aneffective amount of CAR-expressing immune effector cells, alone or incombination with one or more therapeutic agents. Thus, theCAR-expressing immune effector cell compositions may be administeredalone or in combination with other known cancer treatments, such asradiation therapy, chemotherapy, transplantation, immunotherapy, hormonetherapy, photodynamic therapy, etc. The compositions may also beadministered in combination with antibiotics. Such therapeutic agentsmay be accepted in the art as a standard treatment for a particulardisease state as described herein, such as a particular cancer.Exemplary therapeutic agents contemplated include cytokines, growthfactors, steroids, NSAIDs, DMARDs, anti-inflammatories,chemotherapeutics, radiotherapeutics, therapeutic antibodies, or otheractive and ancillary agents.

In certain embodiments, compositions comprising CAR-expressing immuneeffector cells disclosed herein may be administered in conjunction withany number of chemotherapeutic agents. Illustrative examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on cancers such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A variety of other therapeutic agents may be used in conjunction withthe compositions described herein. In one embodiment, the compositioncomprising CAR-expressing immune effector cells is administered with ananti-inflammatory agent. Anti-inflammatory agents or drugs include, butare not limited to, steroids and glucocorticoids (includingbetamethasone, budesonide, dexamethasone, hydrocortisone acetate,hydrocortisone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs(NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide andmycophenolate.

Other exemplary NSAIDs are chosen from the group consisting ofibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX®(rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplaryanalgesics are chosen from the group consisting of acetaminophen,oxycodone, tramadol of proporxyphene hydrochloride. Exemplaryglucocorticoids are chosen from the group consisting of cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone, orprednisone. Exemplary biological response modifiers include moleculesdirected against cell surface markers (e.g., CD4, CD5, etc.), cytokineinhibitors, such as the TNF antagonists (e.g., etanercept (ENBREL®),adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitorsand adhesion molecule inhibitors. The biological response modifiersinclude monoclonal antibodies as well as recombinant forms of molecules.Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine,methotrexate, penicillamine, leflunomide, sulfasalazine,hydroxychloroquine, Gold (oral (auranofin) and intramuscular) andminocycline.

Illustrative examples of therapeutic antibodies suitable for combinationwith the CAR modified T cells contemplated herein, include but are notlimited to, bavituximab, bevacizumab (avastin), bivatuzumab,blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab,dalotuzumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab, indatuximab,inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab,ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab, andublituximab.

In certain embodiments, the compositions described herein areadministered in conjunction with a cytokine. By “cytokine” as usedherein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;IL-15, IL-21, a tumor necrosis factor such as TNF-alpha or TNF-beta; andother polypeptide factors including LIF and kit ligand (KL). As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In particular embodiments, a composition comprises CAR T cellscontemplated herein that are cultured in the presence of a PI3Kinhibitor as disclosed herein and express one or more of the followingmarkers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DRcan be further isolated by positive or negative selection techniques. Inone embodiment, a composition comprises a specific subpopulation of Tcells, expressing one or more of the markers selected from the groupconsisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; and CD38 orCD62L, CD127, CD197, and CD3ε, is further isolated by positive ornegative selection techniques. In various embodiments, compositions donot express or do not substantially express one or more of the followingmarkers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD62L, CD127, CD197, and CD38 is increasedat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 25 fold, or more compared to apopulation of T cells activated and expanded without a PI3K inhibitor.

In one embodiment, expression of one or more of the markers selectedfrom the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, andLAG3 is decreased at least 1.5 fold, at least 2 fold, at least 3 fold,at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 25 fold, ormore compared to a population of T cells activated and expanded with aPI3K inhibitor.

J. Therapeutic Methods

The genetically modified immune effector cells contemplated hereinprovide improved methods of adoptive immunotherapy for use in thetreatment of B cell related conditions that include, but are not limitedto immunoregulatory conditions and hematological malignancies.

In particular embodiments, the specificity of a primary immune effectorcell is redirected to B cells by genetically modifying the primaryimmune effector cell with a CAR contemplated herein. In variousembodiments, a viral vector is used to genetically modify an immuneeffector cell with a particular polynucleotide encoding a CAR comprisinga murine anti-BCMA antigen binding domain that binds a BCMA polypeptide;a hinge domain; a transmembrane (TM) domain, a short oligo- orpolypeptide linker, that links the TM domain to the intracellularsignaling domain of the CAR; and one or more intracellularco-stimulatory signaling domains; and a primary signaling domain.

In one embodiment, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR thattargets BCMA expressing B cells. In another embodiment, anti-BCMA CAR Tcells are cultured in the presence of IL-2 and a PI3K inhibitor toincrease the therapeutic properties and persistence of the CAR T cells.The CAR T cell are then infused to a recipient in need thereof. Theinfused cell is able to kill disease causing B cells in the recipient.Unlike antibody therapies, CAR T cells are able to replicate in vivoresulting in long-term persistence that can lead to sustained cancertherapy.

In one embodiment, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth.

In particular embodiments, compositions comprising immune effector cellscomprising the CARs contemplated herein are used in the treatment ofconditions associated with abnormal B cell activity.

Illustrative examples of conditions that can be treated, prevented orameliorated using the immune effector cells comprising the CARscontemplated herein include, but are not limited to: systemic lupuserythematosus, rheumatoid arthritis, myasthenia gravis, autoimmunehemolytic anemia, idiopathic thrombocytopenia purpura, anti-phospholipidsyndrome, Chagas' disease, Grave's disease, Wegener's granulomatosis,poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris,scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCAassociated vasculitis, Goodpasture's disease, Kawasaki disease, andrapidly progressive glomerulonephritis.

The modified immune effector cells may also have application in plasmacell disorders such as heavy-chain disease, primary orimmunocyte-associated amyloidosis, and monoclonal gammopathy ofundetermined significance (MGUS).

As use herein, “B cell malignancy” refers to a type of cancer that formsin B cells (a type of immune system cell) as discussed infra.

In particular embodiments, compositions comprising CAR-modified T cellscontemplated herein are used in the treatment of hematologicmalignancies, including but not limited to B cell malignancies such as,for example, multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL).

Multiple myeloma is a B cell malignancy of mature plasma cell morphologycharacterized by the neoplastic transformation of a single clone ofthese types of cells. These plasma cells proliferate in BM and mayinvade adjacent bone and sometimes the blood. Variant forms of multiplemyeloma include overt multiple myeloma, smoldering multiple myeloma,plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteoscleroticmyeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma(see, for example, Braunwald, et al. (eds), Harrison's Principles ofInternal Medicine, 15th Edition (McGraw-Hill 2001)).

Non-Hodgkin lymphoma encompasses a large group of cancers of lymphocytes(white blood cells). Non-Hodgkin lymphomas can occur at any age and areoften marked by lymph nodes that are larger than normal, fever, andweight loss. There are many different types of non-Hodgkin lymphoma. Forexample, non-Hodgkin's lymphoma can be divided into aggressive(fast-growing) and indolent (slow-growing) types. Although non-Hodgkinlymphomas can be derived from B cells and T-cells, as used herein, theterm “non-Hodgkin lymphoma” and “B cell non-Hodgkin lymphoma” are usedinterchangeably. B cell non-Hodgkin lymphomas (NHL) include Burkittlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), diffuse large B cell lymphoma, follicular lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,and mantle cell lymphoma. Lymphomas that occur after bone marrow or stemcell transplantation are usually B cell non-Hodgkin lymphomas.

Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancerthat causes a slow increase in immature white blood cells called Blymphocytes, or B cells. Cancer cells spread through the blood and bonemarrow, and can also affect the lymph nodes or other organs such as theliver and spleen. CLL eventually causes the bone marrow to fail.Sometimes, in later stages of the disease, the disease is called smalllymphocytic lymphoma.

In particular embodiments, methods comprising administering atherapeutically effective amount of CAR-expressing immune effector cellscontemplated herein or a composition comprising the same, to a patientin need thereof, alone or in combination with one or more therapeuticagents, are provided. In certain embodiments, the cells of the inventionare used in the treatment of patients at risk for developing a conditionassociated with abnormal B cell activity or a B cell malignancy. Thus,the present invention provides methods for the treatment or preventionof a condition associated with abnormal B cell activity or a B cellmalignancy comprising administering to a subject in need thereof, atherapeutically effective amount of the CAR-modified cells contemplatedherein.

As used herein, the terms “individual” and “subject” are often usedinterchangeably and refer to any animal that exhibits a symptom of adisease, disorder, or condition that can be treated with the genetherapy vectors, cell-based therapeutics, and methods disclosedelsewhere herein. In preferred embodiments, a subject includes anyanimal that exhibits symptoms of a disease, disorder, or condition ofthe hematopoietic system, e.g., a B cell malignancy, that can be treatedwith the gene therapy vectors, cell-based therapeutics, and methodsdisclosed elsewhere herein. Suitable subjects (e.g., patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, preferably, human patients, are included. Typical subjectsinclude human patients that have a B cell malignancy, have beendiagnosed with a B cell malignancy, or are at risk or having a B cellmalignancy.

As used herein, the term “patient” refers to a subject that has beendiagnosed with a particular disease, disorder, or condition that can betreated with the gene therapy vectors, cell-based therapeutics, andmethods disclosed elsewhere herein.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition, and may include even minimal reductions in oneor more measurable markers of the disease or condition being treated.Treatment can involve optionally either the reduction or amelioration ofsymptoms of the disease or condition, or the delaying of the progressionof the disease or condition. “Treatment” does not necessarily indicatecomplete eradication or cure of the disease or condition, or associatedsymptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition. It also refers to delaying the onset or recurrence of adisease or condition or delaying the occurrence or recurrence of thesymptoms of a disease or condition. As used herein, “prevention” andsimilar words also includes reducing the intensity, effect, symptomsand/or burden of a disease or condition prior to onset or recurrence ofthe disease or condition.

By “enhance” or “promote,” or “increase” or “expand” refers generally tothe ability of a composition contemplated herein, e.g., a geneticallymodified T cell or vector encoding a CAR, to produce, elicit, or cause agreater physiological response (i.e., downstream effects) compared tothe response caused by either vehicle or a control molecule/composition.A measurable physiological response may include an increase in T cellexpansion, activation, persistence, and/or an increase in cancer cellkilling ability, among others apparent from the understanding in the artand the description herein. An “increased” or “enhanced” amount istypically a “statistically significant” amount, and may include anincrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30or more times (e.g., 500, 1000 times) (including all integers anddecimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.)the response produced by vehicle or a control composition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of composition contemplated herein to produce,elicit, or cause a lesser physiological response (i.e., downstreameffects) compared to the response caused by either vehicle or a controlmolecule/composition. A “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include an decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response(reference response) produced by vehicle, a control composition, or theresponse in a particular cell lineage.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” or “no substantial decrease” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a lesser physiological response (i.e., downstream effects) in acell, as compared to the response caused by either vehicle, a controlmolecule/composition, or the response in a particular cell lineage. Acomparable response is one that is not significantly different ormeasurable different from the reference response.

In one embodiment, a method of treating a B cell related condition in asubject in need thereof comprises administering an effective amount,e.g., therapeutically effective amount of a composition comprisinggenetically modified immune effector cells contemplated herein. Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the amount of T cells in the composition administeredto a subject is at least 0.1×10⁵ cells, at least 0.5×10⁵ cells, at least1×10⁵ cells, at least 5×10⁵ cells, at least 1×10⁶ cells, at least0.5×10⁷ cells, at least 1×10⁷ cells, at least 0.5×10⁸ cells, at least1×10⁸ cells, at least 0.5×10⁹ cells, at least 1×10⁹ cells, at least2×10⁹ cells, at least 3×10⁹ cells, at least 4×10⁹ cells, at least 5×10⁹cells, or at least 1×10¹⁰ cells. In particular embodiments, about 1×10⁷CAR T cells to about 1×10⁹ CART cells, about 2×10⁷ CART cells to about0.9×10⁹ CART cells, about 3×10⁷ CART cells to about 0.8×10⁹ CART cells,about 4×10⁷ CART cells to about 0.7×10⁹ CART cells, about 5×10⁷ CARTcells to about 0.6×10⁹ CAR T cells, or about 5×10⁷ CART cells to about0.5×10⁹ CAR T cells are administered to a subject.

In one embodiment, the amount of T cells in the composition administeredto a subject is at least 0.1×10⁴ cells/kg of bodyweight, at least0.5×10⁴ cells/kg of bodyweight, at least 1×10⁴ cells/kg of bodyweight,at least 5×10⁴ cells/kg of bodyweight, at least 1×10⁵ cells/kg ofbodyweight, at least 0.5×10⁶ cells/kg of bodyweight, at least 1×10⁶cells/kg of bodyweight, at least 0.5×10⁷ cells/kg of bodyweight, atleast 1×10⁷ cells/kg of bodyweight, at least 0.5×10⁸ cells/kg ofbodyweight, at least 1×10⁸ cells/kg of bodyweight, at least 2×10⁸cells/kg of bodyweight, at least 3×10⁸ cells/kg of bodyweight, at least4×10⁸ cells/kg of bodyweight, at least 5×10⁸ cells/kg of bodyweight, orat least 1×10⁹ cells/kg of bodyweight. In particular embodiments, about1×10⁶ CAR T cells/kg of bodyweight to about 1×10⁸ CAR T cells/kg ofbodyweight, about 2×10⁶ CART cells/kg of bodyweight to about 0.9×10⁸CART cells/kg of bodyweight, about 3×10⁶ CAR T cells/kg of bodyweight toabout 0.8×10⁸ CART cells/kg of bodyweight, about 4×10⁶ CART cells/kg ofbodyweight to about 0.7×10⁸ CAR T cells/kg of bodyweight, about 5×10⁶CART cells/kg of bodyweight to about 0.6×10⁸ CART cells/kg ofbodyweight, or about 5×10⁶ CART cells/kg of bodyweight to about 0.5×10⁸CART cells/kg of bodyweight are administered to a subject.

One of ordinary skill in the art would recognize that multipleadministrations of the compositions of the invention may be required toeffect the desired therapy. For example a composition may beadministered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a spanof 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

In certain embodiments, it may be desirable to administer activatedimmune effector cells to a subject and then subsequently redraw blood(or have an apheresis performed), activate immune effector cellstherefrom according to the present invention, and reinfuse the patientwith these activated and expanded immune effector cells. This processcan be carried out multiple times every few weeks. In certainembodiments, immune effector cells can be activated from blood draws offrom 10 cc to 400 cc. In certain embodiments, immune effector cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, 100 cc, 150 cc, 200 cc, 250 cc, 300 cc, 350 cc, or 400 ccor more. Not to be bound by theory, using this multiple blooddraw/multiple reinfusion protocol may serve to select out certainpopulations of immune effector cells.

The administration of the compositions contemplated herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. In apreferred embodiment, compositions are administered parenterally. Thephrases “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravascular, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intratumoral, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. In one embodiment, the compositions contemplatedherein are administered to a subject by direct injection into a tumor,lymph node, or site of infection.

In one embodiment, a subject in need thereof is administered aneffective amount of a composition to increase a cellular immune responseto a B cell related condition in the subject. The immune response mayinclude cellular immune responses mediated by cytotoxic T cells capableof killing infected cells, regulatory T cells, and helper T cellresponses. Humoral immune responses, mediated primarily by helper Tcells capable of activating B cells thus leading to antibody production,may also be induced. A variety of techniques may be used for analyzingthe type of immune responses induced by the compositions of the presentinvention, which are well described in the art; e.g., Current Protocolsin Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H.Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons,NY, N.Y.

In the case of T cell-mediated killing, CAR-ligand binding initiates CARsignaling to the T cell, resulting in activation of a variety of T cellsignaling pathways that induce the T cell to produce or release proteinscapable of inducing target cell apoptosis by various mechanisms. These Tcell-mediated mechanisms include (but are not limited to) the transferof intracellular cytotoxic granules from the T cell into the targetcell, T cell secretion of pro-inflammatory cytokines that can inducetarget cell killing directly (or indirectly via recruitment of otherkiller effector cells), and up regulation of death receptor ligands(e.g. FasL) on the T cell surface that induce target cell apoptosisfollowing binding to their cognate death receptor (e.g. Fas) on thetarget cell.

In one embodiment, the invention provides a method of treating a subjectdiagnosed with a B cell related condition comprising removing immuneeffector cells from a subject diagnosed with a BCMA-expressing B cellrelated condition, genetically modifying said immune effector cells witha vector comprising a nucleic acid encoding a CAR as contemplatedherein, thereby producing a population of modified immune effectorcells, and administering the population of modified immune effectorcells to the same subject. In a preferred embodiment, the immuneeffector cells comprise T cells.

In certain embodiments, the present invention also provides methods forstimulating an immune effector cell mediated immune modulator responseto a target cell population in a subject comprising the steps ofadministering to the subject an immune effector cell populationexpressing a nucleic acid construct encoding a CAR molecule.

The methods for administering the cell compositions described hereinincludes any method which is effective to result in reintroduction of exvivo genetically modified immune effector cells that either directlyexpress a CAR of the invention in the subject or on reintroduction ofthe genetically modified progenitors of immune effector cells that onintroduction into a subject differentiate into mature immune effectorcells that express the CAR. One method comprises transducing peripheralblood T cells ex vivo with a nucleic acid construct in accordance withthe invention and returning the transduced cells into the subject.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1 Construction of BCMA CARs

CARs containing murine anti-BCMA scFv antibodies were designed tocontain an MND promoter operably linked to anti-BMCA scFv, a hinge andtransmembrane domain from CD8α and a CD137 co-stimulatory domainsfollowed by the intracellular signaling domain of the CD3ζ chain. See,e.g., FIG. 1. The BCMA CAR shown in FIG. 1 comprises a CD8α signalpeptide (SP) sequence for the surface expression on immune effectorcells. The polynucleotide sequence of an exemplary BCMA CAR is set forthin SEQ ID NO: 10; an exemplary polypeptide sequences of a BCMA CAR isset forth in SEQ ID NO: 9; and a vector map of an exemplary CARconstruct is shown in FIG. 1. Table 3 shows the Identity, GenbankReference, Source Name and Citation for the various nucleotide segmentsof an BCMA CAR lentiviral vector.

TABLE 3 Nucleotides Identity GenBank Reference Source Name Citation  1-185 pUC19 plasmid Accession #L09137.2 pUC19 New England backbone nt1-185 Biolabs 185-222 Linker Not applicable Synthetic Not applicable223-800 CMV Not Applicable pHCMV (1994) PNAS 91:9564-68  801-1136 R, U5,PBS, and Accession #M19921.2 pNL4-3 Maldarelli, et.al. packagingsequences nt 454-789 (1991) J Virol: 65(11):5732-43 1137-1139 Gag startcodon Not Applicable Synthetic Not applicable (ATG) changed to stopcodon (TAG) 1140-1240 HIV-1 gag sequence Accession #M19921.2 pNL4-3Maldarelli, et.al. nt 793-893 (1991) J Virol: 65(11):5732-43 1241-1243HIV-1 gag sequence Not Applicable Synthetic Not applicable changed to asecond stop codon 1244-1595 HIV-1 gag sequence Accession #M19921.2pNL4-3 Maldarelli, et.al. nt 897-1248 (1991) J Virol: 65(11):5732-431596-1992 HIV-1 pol Accession #M19921.2 pNL4-3 Maldarelli, et.al.cPPT/CTS nt 4745-5125 (1991) J Virol: 65(11):5732-43 1993-2517 HIV-1,isolate HXB3 Accession #M14100.1 PgTAT-CMV Malim, M.H. env region (RRE)nt 1875-2399 Nature (1988) 335:181-183 2518-2693 HIV-1 env Accession#M19921.2 pNL4-3 Maldarelli, et.al. sequences nt 8290-8470 (1991) S/A JVirol: 65(11):5732-43 2694-2708 Linker Not applicable Synthetic Notapplicable 2709-3096 MND Not applicable pccl-c- Challita et al.MNDU3c-x2 (1995) J.Virol. 69:748-755 3097-3124 Linker Not applicableSynthetic Not applicable 3125-3187 Signal peptide Accession # CD8asignal Not applicable NM_001768 peptide 3188-3934 BCMA02 scFv Notapplicable Synthetic Not applicable (V_(L)1-linker-V_(H)0) 3935-4141CD8a Accession # CD8a hinge Milone et al hinge and TM NM_001768 and TM(2009) Mol Ther 17(8):1453-64 4144-4269 CD137 (4-1BB) Accession # CD137Milone et al signaling domain NM_001561 signaling (2009) domain Mol Ther17(8):1453-64 4270-4606 CD3-ζ signaling Accession # CD3-ζ Milone et aldomain NM_000734 signaling (2009) domain Mol Ther 17(8):1453-644607-4717 HIV-1 ppt and Accession #M19921.2 pNL4-3 Maldarelli, et.al.part of 3’ U3 nt 9005-9110 (1991) J Virol: 65(11):5732-43 4718-4834HIV-1 part of U3 Accession #M19921.2 pNL4-3 Maldarelli, et.al. (399bpdeletion) nt 9511-9627 (1991) and R J Virol: 65(11):5732-43 4835-4858Synthetic polyA Not applicable Synthetic Levitt, N. Genes & Dev (1989)3:1019-1025 4859-4877 Linker Not applicable Synthetic Not Applicable4878-7350 pUC19 backbone Accession #L09137.2 pUC19 New England nt2636-2686 Biolabs

Example 2 Evaluation of a Murine BCMA CAR

Introduction

Adoptive transfer of T cells genetically engineered with chimericantigen receptors (CAR) has emerged as a promising approach to treatcancers. A CAR is an artificial molecule comprised of an antigenreactive single chain variable fragment (scFv) fused to T cell signalingdomains via a transmembrane region. In this example, a CAR moleculespecific to B cell maturation antigen (BCMA) was evaluated. BCMA isexpressed on multiple myeloma, plasmacytoma, and some lymphomas yetnormal expression is limited to plasma cells (Avery et al., 2003;Carpenito et al., 2009; Chiu et al., 2007).

Anti-BCMA02 CAR was constructed using sequences from a mouse anti-BCMAantibody (C11D5.3). Anti-BCMA10 CAR was constructed using modifiedsequences and is a “humanized” version of anti-BCMA02 CAR. In a seriesof in vitro assays, anti-BCMA02 CART cells and anti-BCMA10 CAR T cellsboth exhibited tumor specificity, high CAR expression, and caused potentreactivity to antigen expressing targets. Anti-BCMA02 CAR T cells andanti-BCMA10 CAR T cells were shown to have comparable reactivity toBCMA-expressing tumor cell lines. Although both anti-BCMA02 CAR T cellsand anti-BCMA10 CAR T cells were capable of causing regressions in amouse tumor model, anti-BCMA10 CAR T cells displayed antigen-independentinflammatory cytokine secretion, and thus, have the potential to causeclinical toxicities associated high cytokine levels.

Results

Tonic Inflammatory Cytokine Release from Anti-BCMA10 T Cells Associatedwith Apoptosis

BCMA protein is detectable in the serum of patients with multiplemyeloma (Sanchez et al., 2012). Average serum BCMA in multiple myelomapatients was 10 ng/mL but peaked at levels up to 100 ng/mL. The impactof physiological soluble BCMA levels on the anti-BCMA CAR T cellcandidates was evaluated.

IFNγ release from anti-BCMA02 CART cells, anti-BCMA10 CART cells, andCAR19Δ T cells was examined after a 24 hour culture with soluble BCMA(FIG. 2a ). Anti-BCMA02 CAR T cells responded with minimal cytokinerelease after 24 hour culture with up to 1 ug/mL BCMA. In contrast,anti-BCMA10 CART cells responded with increasing levels of IFNγ thatwere proportional to the concentration of soluble BCMA added to theculture. At 100 ng/mL BCMA, the maximum levels reported in multiplemyeloma patients, anti-BCMA10 CART cells secreted 82.1 ng/ml IFNγcompared to 28.8 ng/ml IFNγ secreted by anti-BCMA02 CAR T cells. IFNγwas even detected in several co-culture experiments with anti-BCMA10 CART cells plus control cell lines that lacked BCMA antigen (FIG. 2b , K562co-culture). These data suggested that anti-BCMA10 CAR T cells hadincreased sensitivity to stimulation by soluble BCMA and the potentialfor antigen-independent cytokine responses in T cells.

The potential of tonic cytokine secretion from anti-BCMA02 CAR T cells,anti-BCMA10 CART cells (10 days from culture initiation), and CAR19Δ Tcells was examined. After manufacture of CAR T cells, growth media fromanti-BCMA02 CAR T cell, anti-BCMA10 CART cell, and CAR19Δ T cellcultures were analyzed for the presence of inflammatory cytokines.Despite the absence of antigen stimulation, anti-BCMA10 CAR T cellcultures contained greater than 10 ng/mL IFNγ compared to less than 1ng/mL of IFNγ in anti-BCMA02 CAR T cell cultures (FIG. 3). Anti-BCMA10CAR T cell cultures also contained significantly (p<0.001) more TNFα. Tofurther quantify the amount of cytokine produced by anti-BCMA10 CARTcells without antigen stimulation, cytokine release was measured from5×10⁴ CAR T cells during a 24 hour culture. anti-BCMA10 CART cellsproduced significantly higher amounts of inflammatory cytokines MIP1αIFNγ, GMCSF, MIP1β, IL-8, and TNFα compared to anti-BCMA02 CART cells(FIG. 4, p<0.0001). MIP1α and IFNγ concentrations were the highest amongall cytokines examined. Anti-BCMA10 CAR T cells produced 4.7 ng MIP1α5×10⁴ cells/24 hours, 3.0 ng IFNγ/5×10⁴ cells/24 hours and ˜1 ng/5×10⁴cells/24 hours or less of the other cytokines. No significantdifferences in the anti-inflammatory cytokines IL-10, IL-2, and IL-4were detected.

The expression of phenotypic markers of T cell activation at the end ofanti-BCMA10 CAR T cell manufacturing were measure to examine whethertonic inflammatory cytokine secretion was indicative of a hyperactivestate in anti-BCMA10 CAR T cells. HLA-DR and CD25 are surface markersthat normally exhibit peak expression 12-24 hours after T cellactivation and then diminish with time. CAR T cells prepared from threenormal donors showed that an average 40±2% of anti-BCMA02 CAR T cellsexpressed HLA-DR. The expression of HLA-DR in these cells was comparableto untransduced (43±2.3%) T cells and CAR19Δ (32±2.2%) control T cells.In contrast, 88±1.2% anti-BCMA10 CAR T cells expressed HLA-DR (FIG. 5).Expression of another activation marker CD25 was also higher onanti-BCMA10 CAR T cells compared to anti-BCMA02 CAR T cells (53±0.9% vs35±2.4%). Therefore, anti-BCMA10 CART cells exhibited phenotypiccharacteristics of activated T cells in the absence of added antigens.

Hyperactivity in T cells is often associated with activation-inducedcell death (AICD) by apoptosis. Levels of activated caspase-3 weremeasured to examine whether hyperactivity of anti-BCMA10 CAR T cellscould result in higher apoptotic levels compared to anti-BCMA02 CARTcells. 48% of anti-BCMA10 CART cells from two donors had activecaspase-3 compared to 16% of anti-BCMA02 CAR T cells (FIG. 6). Thus, inthe absence of added BCMA antigen, anti-BCMA10 CART cells contain ahigher frequency of apoptotic cells associated with increased activationand inflammatory cytokine secretion compared to anti-BCMA02 CAR T cells.

anti-BCMA02 CART cells and anti-BCMA10 CAR T cells were evaluated forwhether the CAR T cells could selectively respond to low BCMA levels orbe cross reactive to an unrelated antigen in the human serum used for Tcell growth. T anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells weremaintained in media lacking human serum for two days and then switchedinto media containing fetal bovine serum (FBS), human serum (HABS), orHABS in the presence or absence of 100 ng/mL soluble BCMA (FIG. 7). IFNγrelease was assayed 24 hours later by ELISA. Both anti-BCMA02 CART cellsand anti-BCMA10 CAR T cells responded to soluble BCMA. However,anti-BCMA10 CAR T cells secreted 10-times more IFNγ than anti-BCMA02 CART cells. In the absence of BCMA, only anti-BCMA10 CAR T cells releasedIFNγ regardless of culture in fetal bovine serum (FBS)(p=0.0002) orhuman AB serum (HABS)(p=0.0007). These data suggested that inflammatorycytokine secretion was intrinsic to anti-BCMA10 CAR T cells.

Inferior Anti-tumor Function of Anti-BCMA10 CAR T Cells in Mouse Modelof Multiple Myeloma

Hyperactivation and increased apoptosis could negatively impact CAR Tcell persistence in patients and ultimately clinical efficacy. Theanti-tumor function of anti-BCMA02 CAR T cells and anti-BCMA10 CARTcells was examined in a mouse tumor model. NOD scid gamma (NSG) micewith ˜100 mm³ experimental sub-cutaneous human multiple myeloma(RPMI-8226) tumors were treated with 10⁷ anti-BCMA02 CAR T cells, 10⁷anti-BCMA10 CAR T cells, or Bortezomib (velcade). RPMI-8226 growth wasmonitored with calipers. In two independent experiments (FIGS. 8a and 8b), Bortezomib controlled tumor growth compared to vehicle controlanimals. Animals adoptively transferred with anti-BCMA02 CAR T cellsexhibited rapid and durable tumor clearance (inset graphs magnify earlytumor regressions). Adoptive transfer of anti-BCMA10 CART cells alsocaused tumor regressions but was delayed in both experiments compared toanti-BCMA02 CAR T cells.

CONCLUSIONS

anti-BCMA02 CAR T cells and anti-BCMA10 CAR T cells exhibited comparableantitumor function in in vitro assays, but anti-BCMA10 CART cells hadcharacteristics that could negatively impact safety and efficacy inpatient treatment. Anti-BCMA10 CART cells responded robustly withinflammatory cytokine secretion after exposure to physiological levelsof BCMA protein. Cytokine storm or cytokine release syndrome is a knownclinical toxicity associated with CAR T cell therapies. Concerns overcytokine release to BCMA were worsened after observation of tonicactivity of anti-BCMA10 CAR T cells. Even in the absence ofantigen-stimulation, anti-BCMA10 CART cells released high levels ofinflammatory cytokines. Persistent cytokine secretion has the potentialto cause substantial clinical toxicities as well as negatively impactanti-tumor function. Indeed, we found higher composition of apoptoticcells and inferior anti-tumor function in anti-BCMA10 CAR T cellscompared to anti-BCMA02 CAR T cell cultures in a mouse model of multiplemyeloma.

REFERENCES

Avery et al., (2003). BAFF selectively enhances the survival ofplasmablasts generated from human memory B cells. J Clin Invest, 112(2),286-297.

Carpenito et al., (2009). Control of large, established tumor xenograftswith genetically retargeted human T cells containing CD28 and CD137domains. Proc Natl Acad Sci USA, 106(9), 3360-3365.

Chiu et al., (2007). Hodgkin lymphoma cells express TACI and BCMAreceptors and generate survival and proliferation signals in response toBAFF and APRIL. Blood, 109(2), 729-739.

Sanchez et al. (2012). Serum B-cell maturation antigen is elevated inmultiple myeloma and correlates with disease status and survival. Br JHaematol, 158(6), 727-738.

Example 3 Minimal BCMA Expression on Lymphomas Activates Anti-BCMA CAR TCells

The level of BCMA expression on lymphoma and leukemia cell lines (Daudiand Raji) was measured in order to determine if the expression wassufficient to activate anti-BCMA02 CAR T cells.

BCMA expression on lymphoma, leukemia, and multiple myeloma cells wasquantitated using flow cytometry. In this assay, the relative BCMAexpression on the cells was assessed by correlating the fluorescenceintensity of BCMA expression to a known number of bound antibodies(antibody binding capacity, ABC). BCMA expression levels in the lymphomacell lines were compared to BCMA expression levels a multiple myelomacell line (RPMI-8226) known to activate anti-BCMA02 CAR T cells.12590±1275 BCMA02 molecules were expressed on the surface of RPMI-8226cells. By contrast, Daudi cells expressed 1173±234 BCMA02 molecules andJeKo-1 cells (a Mantle cell lymphoma cell line) expressed only 222±138BCMA02 molecules (FIG. 9, circles).

In another set of experiments the activity of anti-BCMA02 CAR T cells tothe minute levels of BCMA observed on lymphoma and leukemia cell lineswas tested (FIG. 9, boxes). Anti-BCMA02 CAR T cells were generated usingstandard methods and activity was assessed by IFNγ ELISA afterco-culture with BCMA-positive and BCMA-negative tumor cell lines.Reactivity of anti-BCMA02 CART cells correlated with the relative amountof BCMA mRNA expression (above a threshold) and/or the density of theBCMA receptor on the surface of various tumor cell lines afterco-culture (FIG. 9). Little, if any, IFNγ is released upon co-culture ofBCMA CAR T cells with BCMA-negative (BCMA-) tumor cell lines:myelogenous leukemia (K562), acute lymphoblastic leukemia (NALM-6 andNALM-16); Mantle cell lymphoma (REC-1); or Hodgkin's lymphoma (HDLM-2).In contrast, substantial amounts of IFNγ was released upon co-culture ofBCMA02 CAR T cells with BCMA-positive (BCMA+) tumor cell lines: B cellchronic lymphoblastic leukemia (MEC-1), Mantle cell lymphoma (JeKo-1),Hodgkin's lymphoma (RPMI-6666), Burkitt's lymphoma (Daudi cells andRamos cells), and multiple myeloma (RPMI-8226).

The reactivity of anti-BCMA02 CART cells to BCMA expressing Burkitt'slymphoma cells (Daudi cells) extended to in vivo animal studies. Daudicells also express CD19. The in vivo activity of anti-BCMA02 CART cellswas compared to the in vivo activity of anti-CD19 CART cells. NOD scidgamma (NSG) mice were injected IV with 2×10⁶ Daudi cells and allowed toaccumulate a large systemic tumor burden before being treated with CAR Tcells. CAR T cells were administered at 8 days and 18 days post-tumorinduction (FIGS. 10A and 10B, respectively). The vehicle and negativecontrol (anti-CD19Δ CAR T cells) failed to prevent tumor growth, asshown by log-phase increases in bioluminescence, resulting in weightloss and death (FIG. 10A, leftmost two mouse panels). Anti-CD19 andanti-BCMA02 CAR T cells prevented tumor growth, resulting in maintenanceof body weight and survival. Anti-CD19 and anti-BCMA02 CAR T cells wereequally effective when administered on Day 8 (FIG. 10A, rightmost twomouse panels). Anti-BCMA02 CAR T cells were also effective in decreasingtumor burden when administered at 18 days post-tumor induction. FIG.10B, rightmost panel.

Example 4 Potent In Vitro Activity of Anti-BCMA CAR T Cells

Potent in vitro activity of anti-BCMA02 CAR T cells was achieved with a50 percent reduction anti-BCMA02 CAR expression. T cell populations weretransduced with between 4×10⁸ and 5×10⁷ transducing units of alentivirus encoding an anti-BCM02A CAR molecule. The resulting T cellpopulations showed reduced anti-BCMA02 CAR T cell frequency (assayed aspercent positive) and reduced expression of anti-BCMA02 CAR molecules(assayed as mean florescence intensity:MFI).

The impact of reduced CAR molecule expression on anti-BCMA02 activitywas determined. The frequency of anti-BCMA CAR-positive T cells wasnormalized with untransduced T cells to contain 26±4% BCMA-reactive Tcells (FIG. 11A). MFI of the normalized anti-BCMA02 CART cells rangedfrom 885 to 1875 (FIG. 11B). K562 is a CML cell line that lacks BCMAexpression. K562 cells were engineered to express BCMA and were used inan in vitro cytolytic assay to assess activity of anti-BCMA02 CAR Tcells with varied BCMA CAR expression (FIG. 11C). K562 cells werelabeled with cell trace violet while K562 cells stably expressing BCMA(K562-BCMA) were labeled with CFSE. T cells, K562 cells, and K562-BCMAcells were harvested, washed, and resuspended in media lacking exogenouscytokines. Cells were cultured at a 20:1 or 10:1 effector (E; T cell) totarget (T; 1:1 mix of K562 and K562 BCMA cells) ratio for 4 h in a 37°C., 5% CO₂ incubator. Cells were then stained with Live/Dead andanalyzed by FACS. Cytotoxicity was determined by the difference in theratio of K562:K562-BCMA cells normalized to conditions lacking T cells.

Example 5 Anti-BCMA CAR T Cell Manufacturing Process

Unique anti-BCMA02 CAR T cell products are manufactured for each patienttreatment. The reliability of the manufacturing process for anti-BCMA02CAR T cell products was evaluated by generating anti-BCMA02 CAR T cellsfrom 11 individual normal donor PBMC. Anti-BCMA02 CAR T cell expansionfrom each donor was comparable to a matched untransduced cultureperformed in parallel (FIG. 12A).

At the end of the culture period (day 10), T cell transductionefficiency was assessed by quantitating the number of integratedlentiviruses with qPCR and lentiviral-specific primer sets (vector copynumber, VCN). Anti-BCMA02 CAR T cell cultures from the 11 donors showedcomparable lentiviral transduction efficiency (FIG. 12B). The frequencyof anti-BCMA02 CAR positive T cells was measured by flow cytometry andBCMA expression was found to be comparable across all donors (FIG. 12C).

The activity of each anti-BCMA02 CAR T cell product was assessed byIFNγ-release after co-culture with K562 cells engineered to expressBCMA. All anti-BCMA CAR02 T cell products exhibited therapeuticallyrelevant levels of IFNγ release when exposed to BCMA-expressing K562cells (FIG. 12D).

Example 6 CD62L, CD127, CD197, and CD38 Expression on Cart Cells Treatedwith IL-2 OR IL-2 and ZSTK474

CAR T cells cultured with IL-2 and ZSTK474 show increased CD62Lexpression compared to CAR T cells cultured with IL-2 alone. Expressionanalysis of 29 additional cell surface markers on anti-BCMA02 CAR Tcells cultured with IL-2 and ZSTK474 was performed using multiparametermass cytometry (CyTOF) and compared with CART cells cultured in IL-2alone. Three additional markers (CD127, CD197, and CD38) showedincreased expression in the IL-2+ZSTK474 treated CAR T cells compared toCAR T cells treated with IL-2 alone. Thus, co-expression of CD62L,CD127, CD197, and CD38 further stratified ZSTK474-cultured CART cells.After culture in media containing IL-2, 7.44% of anti-BCMA02 CAR Tco-expressed CD127, CD197 and CD38 compared to 24.5% of anti-BCMA02 CARTcells cultured with IL-2 and ZSTK474. The Venn diagram in FIG. 13illustrates the co-expression of CD127, CD197 and CD38 in CD62L positiveanti-BCMA02 T cells.

Example 7 ZSTK474 Treatment Increases the Frequency of CD8 T Cells

CD8 expression was quantified in anti-BCMA02 CAR T cells treated withIL-2 alone or IL-2 and ZSTK474. CD8 expression was determined using afluorescently-labeled anti-CD8 antibody and flow cytometry. Anti-BCMA02CAR T cells from seven normal donors cultured with IL-2 and ZSTK474 hadsignificantly higher CD8 expression compared to anti-BCMA02 CAR T cellscultured with IL-2 alone. FIG. 14.

Example 8 Lack of Antigen-Independent Activity in ZSTK474 TreatedAnti-BCMA CAR T Cells

Tonic activity of CAR T cells in the absence of antigen has beenassociated with reduced biological activity. Tonic activity ofanti-BCMA02 CAR T cells was assessed by quantifying interferon-γ (IFN-γ)release in the absence of antigen after culture in the presence of IL-2and ZSTK474 compared to standard culture conditions with IL-2 alone.Anti-BCMA CAR T cells cultures were prepared using a system directlyscalable to large clinical manufacturing processes. Briefly, peripheralblood mononuclear cells (PBMC) were cultured in static flasks in mediacontaining IL-2 (CellGenix) and antibodies specific for CD3 and CD28(Miltenyi Biotec). 2×10⁸ transducing units of lentivirus encodinganti-BCMA CARs were added one day after culture initiation.

Anti-BCMA02 CAR T cells were maintained in log-phase by adding freshmedia containing IL-2 and an optimized dose of ZSTK474 for a total often days of culture. At the end of manufacture, an equivalent number ofanti-BCMA02 CAR T cells were re-cultured for 24 hours in media alone.The amount of IFN-γ released in 24 hours was quantified by ELISA. Inthis assay IFN-γ levels below 200 pg/mL represent no tonic activity.FIG. 15 shows the amount of IFN-γ released by anti-BCMA02 CAR T cellsfrom 14 donors is consistent with lacking tonic activity whether or notthe CAR T cells are cultured with ZSTK474.

Example 9 ZSTK474 Treated Anti-BCMA02 CAR T Cells Show TherapeuticActivity in a Lymphoma Tumor Model

Daudi tumors were used to interrogate the anti-tumor activity ofanti-BCMA02 CAR T cells cultured with IL-2 or IL-2 and ZSTK474. Daudicells express a low level of BCMA protein and provide an aggressive anddifficult to treat lymphoma tumor model.

2×10⁶ Daudi tumor cells were labeled with a firefly luciferase gene andinjected into NOD scid IL-2 receptor gamma chain knockout mice (NSG) byintravenous injection. After tumors were allowed to form, 1×10⁷ CAR Tcells were injected in to tumor bearing mice. Mice were injected with i)anti-BCMA02 CAR T cells treated for ten days with IL-2 or IL-2 andZSTK474; or ii) a truncated signaling deficient anti-BCMA02 (tBCMA02)CAR T cell treated for ten days with IL-2 and ZSTK474. Tumor growth wasmonitored by bioluminescence using a Xenogen-IVIS Imaging system.

Complete tumor regression was observed in 50% of mice administered theanti-BCMA02 CAR T cells treated with IL-2 and ZSTK474. FIG. 16.

Example 10 ZSTK474 Treated CAR T Cells Show Therapeutic Activity in aMouse Model of Human Myeloma

Animals with 100 mm³ sub cutaneous multiple myeloma tumors (RPMI-8226)were infused with equivalent CAR T cell doses (1×10⁶ anti-BCMA02CAR-positive T cells) or unmodified T cells from a matched T cell donor(untransduced). Anti-BCMA CAR T cells were treated with IL-2 or IL-2 andZSTK474 as described in Example 8.

Animals treated with IL-2- or IL-2 and ZSTK474-cultured anti-BCMA02 CART cells completely prevented tumor outgrowth. FIG. 17. In contrast,animals treated with untransduced or vehicle were unable to controltumor growth. FIG. 17.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

The invention claimed is:
 1. A polynucleotide encoding a chimericantigen receptor (CAR) comprising the polynucleotide sequence set forthin SEQ ID NO:
 10. 2. A vector comprising the polynucleotide of claim 1.3. The vector of claim 2, wherein the vector is an expression vector. 4.The vector of claim 2, wherein the vector is an episomal vector.
 5. Thevector of claim 2, wherein the vector is a viral vector.
 6. The vectorof claim 2, wherein the vector is a retroviral vector.
 7. The vector ofclaim 2, wherein the vector is a lentiviral vector.
 8. The vector ofclaim 2, wherein the lentiviral vector is selected from the groupconsisting essentially of: human immunodeficiency virus 1 (HIV-1); humanimmunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus; caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV).
 9. The vector ofclaim 8, comprising a left (5′) retroviral LTR, a Psi (Ψ ) packagingsignal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviralexport element; a promoter operably linked to the polynucleotideencoding the CAR; and a right (3′) retroviral LTR.
 10. The vector ofclaim 9, further comprising a heterologous polyadenylation sequence. 11.The vector of claim 10, wherein the polyadenylation sequence is a bovinegrowth hormone polyadenylation or signal rabbit β-globin polyadenylationsequence.
 12. The vector of claim 9, wherein the promoter of the 5′ LTRis replaced with a heterologous promoter.
 13. The vector of claim 12,wherein the heterologous promoter is a cytomegalovirus (CMV) promoter, aRous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.14. The vector of claim 9, wherein the 5′ LTR or 3′ LTR is a lentivirusLTR.
 15. The vector of claim 9, wherein the 3′ LTR comprises one or moremodifications.
 16. The vector of claim 9, wherein the 3′ LTR comprisesone or more deletions.
 17. The vector of claim 9, wherein the 3′ LTR isa self-inactivating (SIN) LTR.
 18. The vector of claim 9, wherein thepromoter operably linked to the polynucleotide encoding the CAR isselected from the group consisting of: a cytomegalovirus immediate earlygene promoter (CMV), an elongation factor 1 alpha promoter (EF1-α), aphosphoglycerate kinase-1 promoter (PGK), a ubiquitin-C promoter(UBQ-C), a cytomegalovirus enhancer/chicken beta-actin promoter (CAG),polyoma enhancer/herpes simplex thymidine kinase promoter (MC1), a betaactin promoter (β-ACT), a simian virus 40 promoter (SV40), and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer-binding site substituted (MND) promoter.
 19. Animmune effector cell comprising the vector of any one of claims 2 to 18.20. The immune effector cell of claim 19, wherein the immune effectorcell is selected from the group consisting of: a T lymphocyte and anatural killer (NK) cell.
 21. A composition comprising the immuneeffector cell of claim 20 and a physiologically acceptable excipient.